Protein kinase C inhibitors and uses thereof

ABSTRACT

This disclosure concerns compounds which are useful as inhibitors of protein kinase C (PKC) and are thus useful for treating a variety of diseases and disorders that are mediated or sustained through the activity of PKC. This disclosure also relates to pharmaceutical compositions comprising these compounds, methods of using these compounds in the treatment of various diseases and disorders, processes for preparing these compounds and intermediates useful in these processes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/209,997, filed Mar. 13, 2014, which claims priority under 35 U.S.C.§119(e) to the filing date of U.S. Provisional Application No.61/783,647, filed Mar. 14, 2013, the disclosures of each of which areincorporated herein by reference.

BACKGROUND

Protein kinase C (“PKC”) is a key enzyme in signal transduction involvedin a variety of cellular functions, including cell growth, regulation ofgene expression, and ion channel activity. The PKC family of isozymesincludes at least 11 different protein kinases that can be divided intoat least three subfamilies based on their homology and sensitivity toactivators. Each isozyme includes a number of homologous (“conserved” or“C”) domains interspersed with isozyme-unique (“variable” or “V”)domains. Members of the “classical” or “cPKC” subfamily, PKC α, β_(i),β_(ii) and γ, contain four homologous domains (C1, C2, C3 and C4) andrequire calcium, phosphatidylserine, and diacylglycerol or phorbolesters for activation. Members of the “novel” or “nPKC” subfamily, PKCδ, ε, η and θ, lack the C2 homologous domain and do not require calciumfor activation. Finally, members of the “atypical” or “αPKC” subfamily,PKC ζ and λ/i, lack both the C2 and one-half of the C1 homologousdomains and are insensitive to diacylglycerol, phorbol esters andcalcium.

SUMMARY

This disclosure concerns compounds which are useful as inhibitors ofprotein kinase C (PKC) and are thus useful for treating a variety ofdiseases and disorders that are mediated or sustained through theactivity of PKC. This disclosure also relates to pharmaceuticalcompositions comprising these compounds, methods of using thesecompounds in the treatment of various diseases and disorders, processesfor preparing these compounds and intermediates useful in theseprocesses.

Exemplary chemical structures are provided throughout the disclosure. Byway of example, such compounds are represented by the following formula(I):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

G is halogen or —NY²Ar¹;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl;

or a salt or stereoisomer thereof.

DETAILED DESCRIPTION

This disclosure concerns compounds which are useful as inhibitors ofprotein kinase C (PKC) and are thus useful for treating a variety ofdiseases and disorders that are mediated or sustained through theactivity of PKC. This disclosure also relates to pharmaceuticalcompositions comprising these compounds, methods of using thesecompounds in the treatment of various diseases and disorders, processesfor preparing these compounds and intermediates useful in theseprocesses.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is specifically contemplated. The upper and lower limitsof these smaller ranges may independently be included in the smallerranges, and are also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Except as otherwise noted, the methods and techniques of the presentembodiments are generally performed according to conventional methodswell known in the art and as described in various general and morespecific references that are cited and discussed throughout the presentspecification. See, e.g., Loudon, Organic Chemistry, Fourth Edition, NewYork: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith andMarch, March's Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, Fifth Edition, Wiley-Interscience, 2001; or Vogel, A Textbookof Practical Organic Chemistry, Including Qualitative Organic Analysis,Fourth Edition, New York: Longman, 1978.

The nomenclature used herein to name the subject compounds isillustrated in the Examples herein. This nomenclature has generally beenderived using the commercially-available AutoNom software (MDL, SanLeandro, Calif.).

TERMS

The following terms have the following meanings unless otherwiseindicated. Any undefined terms have their art recognized meanings.

“Alkyl” refers to monovalent saturated aliphatic hydrocarbyl groupshaving from 1 to 10 carbon atoms and preferably 1 to 6 carbon atoms.This term includes, by way of example, linear and branched hydrocarbylgroups such as methyl (CH₃—), ethyl (CH₃CH₂—), n-propyl (CH₃CH₂CH₂—),isopropyl ((CH₃)₂CH—), n-butyl (CH₃CH₂CH₂CH₂—), isobutyl ((CH₃)₂CHCH₂—),sec-butyl ((CH₃)(CH₃CH₂)CH—), t-butyl ((CH₃)₃C—), n-pentyl(CH₃CH₂CH₂CH₂CH₂—), and neopentyl ((CH₃)₃CCH₂—).

The term “substituted alkyl” refers to an alkyl group as defined hereinwherein one or more carbon atoms in the alkyl chain have been optionallyreplaced with a heteroatom such as —O—, —N—, —S—, —S(O)_(n)— (where n is0 to 2), —NR— (where R is hydrogen or alkyl) and having from 1 to 5substituents selected from the group consisting of alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl, —SO₂-aryl,—SO₂-heteroaryl, and —NR^(a)R^(b), wherein R^(a) and R^(b) may be thesame or different and are chosen from hydrogen, optionally substitutedalkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, aryl, heteroaryl andheterocyclic.

“Alkylene” refers to divalent aliphatic hydrocarbyl groups preferablyhaving from 1 to 6 and more preferably 1 to 3 carbon atoms that areeither straight-chained or branched, and which are optionallyinterrupted with one or more groups selected from —O—, —NR¹⁰—,—NR¹⁰C(O)—, —C(O)NR¹⁰— and the like. This term includes, by way ofexample, methylene (—CH₂—), ethylene (—CH₂CH₂—), n-propylene(—CH₂CH₂CH₂—), iso-propylene (—CH₂CH(CH₃)—), (—C(CH₃)₂CH₂CH₂—),(—C(CH₃)₂CH₂C(O)—), (—C(CH₃)₂CH₂C(O)NH—), (—CH(CH₃)CH₂—), and the like.

“Substituted alkylene” refers to an alkylene group having from 1 to 3hydrogens replaced with substituents as described for carbons in thedefinition of “substituted” below.

The term “alkane” refers to alkyl group and alkylene group, as definedherein.

The term “alkylaminoalkyl”, “alkylaminoalkenyl” and “alkylaminoalkynyl”refers to the groups R′NHR″— where R′ is alkyl group as defined hereinand R″ is alkylene, alkenylene or alkynylene group as defined herein.

The term “alkaryl” or “aralkyl” refers to the groups -alkylene-aryl and-substituted alkylene-aryl where alkylene, substituted alkylene and arylare defined herein.

“Alkoxy” refers to the group —O-alkyl, wherein alkyl is as definedherein. Alkoxy includes, by way of example, methoxy, ethoxy, n-propoxy,isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy, and the like. Theterm “alkoxy” also refers to the groups alkenyl-O—, cycloalkyl-O—,cycloalkenyl-O—, and alkynyl-O—, where alkenyl, cycloalkyl,cycloalkenyl, and alkynyl are as defined herein.

The term “substituted alkoxy” refers to the groups substituted alkyl-O—,substituted alkenyl-O—, substituted cycloalkyl-O—, substitutedcycloalkenyl-O—, and substituted alkynyl-O— where substituted alkyl,substituted alkenyl, substituted cycloalkyl, substituted cycloalkenyland substituted alkynyl are as defined herein.

The term “alkoxyamino” refers to the group —NH-alkoxy, wherein alkoxy isdefined herein.

The term “haloalkoxy” refers to the groups alkyl-O— wherein one or morehydrogen atoms on the alkyl group have been substituted with a halogroup and include, by way of examples, groups such as trifluoromethoxy,and the like.

The term “haloalkyl” refers to a substituted alkyl group as describedabove, wherein one or more hydrogen atoms on the alkyl group have beensubstituted with a halo group. Examples of such groups include, withoutlimitation, fluoroalkyl groups, such as trifluoromethyl, difluoromethyl,trifluoroethyl and the like.

The term “alkylalkoxy” refers to the groups -alkylene-O-alkyl,alkylene-O— substituted alkyl, substituted alkylene-O-alkyl, andsubstituted alkylene-O-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

The term “alkylthioalkoxy” refers to the group -alkylene-S-alkyl,alkylene-S-substituted alkyl, substituted alkylene-S-alkyl andsubstituted alkylene-S-substituted alkyl wherein alkyl, substitutedalkyl, alkylene and substituted alkylene are as defined herein.

“Alkenyl” refers to straight chain or branched hydrocarbyl groups havingfrom 2 to 6 carbon atoms and preferably 2 to 4 carbon atoms and havingat least 1 and preferably from 1 to 2 sites of double bond unsaturation.This term includes, by way of example, bi-vinyl, allyl, andbut-3-en-1-yl. Included within this term are the cis and trans isomersor mixtures of these isomers.

The term “substituted alkenyl” refers to an alkenyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Alkynyl” refers to straight or branched monovalent hydrocarbyl groupshaving from 2 to 6 carbon atoms and preferably 2 to 3 carbon atoms andhaving at least 1 and preferably from 1 to 2 sites of triple bondunsaturation. Examples of such alkynyl groups include acetylenyl(—C≡CH), and propargyl (—CH₂C≡CH).

The term “substituted alkynyl” refers to an alkynyl group as definedherein having from 1 to 5 substituents, or from 1 to 3 substituents,selected from alkoxy, substituted alkoxy, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl, and —SO₂-heteroaryl.

“Alkynyloxy” refers to the group —O-alkynyl, wherein alkynyl is asdefined herein. Alkynyloxy includes, by way of example, ethynyloxy,propynyloxy, and the like.

“Acyl” refers to the groups H—C(O)—, alkyl-C(O)—, substitutedalkyl-C(O)—, alkenyl-C(O)—, substituted alkenyl-C(O)—, alkynyl-C(O)—,substituted alkynyl-C(O)—, cycloalkyl-C(O)—, substitutedcycloalkyl-C(O)—, cycloalkenyl-C(O)—, substituted cycloalkenyl-C(O)—,aryl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substitutedheteroaryl-C(O)—, heterocyclyl-C(O)—, and substitutedheterocyclyl-C(O)—, wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein. For example, acylincludes the “acetyl” group CH₃C(O)—

“Acylamino” refers to the groups —NR²⁰C(O)alkyl, —NR²⁰C(O) substitutedalkyl, NR²⁰C(O)cycloalkyl, —NR²⁰C(O) substituted cycloalkyl,—NR²⁰C(O)cycloalkenyl, —NR²⁰C(O) substituted cycloalkenyl,—NR²⁰C(O)alkenyl, —NR²⁰C(O)substituted alkenyl, —NR²⁰C(O)alkynyl,—NR²⁰C(O)substituted alkynyl, —NR²⁰C(O)aryl, —NR²⁰C(O)substituted aryl,—NR²⁰C(O)heteroaryl, —NR²⁰C(O)substituted heteroaryl,—NR²⁰C(O)heterocyclic, and —NR²⁰C(O)substituted heterocyclic, whereinR²⁰ is hydrogen or alkyl and wherein alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonyl” or the term “aminoacyl” refers to the group—C(O)NR²¹R²², wherein R²¹ and R²² independently are selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, alkenyl,substituted alkenyl, alkynyl, substituted alkynyl, aryl, substitutedaryl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic and where R²¹ and R²² are optionally joinedtogether with the nitrogen bound thereto to form a heterocyclic orsubstituted heterocyclic group, and wherein alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, andsubstituted heterocyclic are as defined herein.

“Aminocarbonylamino” refers to the group —NR²¹C(O)NR²²R²³ where R²¹,R²², and R²³ are independently selected from hydrogen, alkyl, aryl orcycloalkyl, or where two R groups are joined to form a heterocyclylgroup.

The term “alkoxycarbonylamino” refers to the group —NRC(O)OR where eachR is independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclyl wherein alkyl, substituted alkyl, aryl, heteroaryl, andheterocyclyl are as defined herein.

The term “acyloxy” refers to the groups alkyl-C(O)O—, substitutedalkyl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—,aryl-C(O)O—, heteroaryl-C(O)O—, and heterocyclyl-C(O)O— wherein alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, aryl, heteroaryl,and heterocyclyl are as defined herein.

“Aminosulfonyl” refers to the group —SO₂NR²¹R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, substituted heterocyclic and where R²¹ and R²²are optionally joined together with the nitrogen bound thereto to form aheterocyclic or substituted heterocyclic group and alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic and substituted heterocyclic are as definedherein.

“Sulfonylamino” refers to the group —NR²¹SO₂R²², wherein R²¹ and R²²independently are selected from the group consisting of hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic and where R²¹ andR²² are optionally joined together with the atoms bound thereto to forma heterocyclic or substituted heterocyclic group, and wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein.

“Aryl” or “Ar” refers to a monovalent aromatic carbocyclic group of from6 to 18 carbon atoms having a single ring (such as is present in aphenyl group) or a ring system having multiple condensed rings (examplesof such aromatic ring systems include naphthyl, anthryl and indanyl)which condensed rings may or may not be aromatic, provided that thepoint of attachment is through an atom of an aromatic ring. This termincludes, by way of example, phenyl and naphthyl. Unless otherwiseconstrained by the definition for the aryl substituent, such aryl groupscan optionally be substituted with from 1 to 5 substituents, or from 1to 3 substituents, selected from acyloxy, hydroxy, thiol, acyl, alkyl,alkoxy, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl,substituted alkoxy, substituted alkenyl, substituted alkynyl,substituted cycloalkyl, substituted cycloalkenyl, amino, substitutedamino, aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl, —SO₂-heteroaryl and trihalomethyl.

“Aryloxy” refers to the group —O-aryl, wherein aryl is as definedherein, including, by way of example, phenoxy, naphthoxy, and the like,including optionally substituted aryl groups as also defined herein.

“Amino” refers to the group —NH₂.

The term “substituted amino” refers to the group —NRR where each R isindependently selected from the group consisting of hydrogen, alkyl,substituted alkyl, cycloalkyl, substituted cycloalkyl, alkenyl,substituted alkenyl, cycloalkenyl, substituted cycloalkenyl, alkynyl,substituted alkynyl, aryl, heteroaryl, and heterocyclyl provided that atleast one R is not hydrogen.

The term “azido” refers to the group —N₃.

“Carboxyl,” “carboxy” or “carboxylate” refers to —CO₂H or salts thereof.

“Carboxyl ester” or “carboxy ester” or the terms “carboxyalkyl” or“carboxylalkyl” refers to the groups —C(O)O-alkyl, —C(O)O-substitutedalkyl, —C(O)O-alkenyl, —C(O)O-substituted alkenyl, —C(O)O-alkynyl,—C(O)O-substituted alkynyl, —C(O)O-aryl, —C(O)O-substituted aryl,—C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-cycloalkenyl,—C(O)O-substituted cycloalkenyl, —C(O)O-heteroaryl, —C(O)O-substitutedheteroaryl, —C(O)O-heterocyclic, and —C(O)O-substituted heterocyclic,wherein alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl,substituted cycloalkenyl, aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclic, and substituted heterocyclic areas defined herein.

“(Carboxyl ester)oxy” or “carbonate” refers to the groups —O—C(O)O—alkyl, —O—C(O)O-substituted alkyl, —O—C(O)O-alkenyl,—O—C(O)O-substituted alkenyl, —O—C(O)O-alkynyl, —O—C(O)O-substitutedalkynyl, —O—C(O)O-aryl, —O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl,—O—C(O)O-substituted cycloalkyl, —O—C(O)O-cycloalkenyl, —O—C(O)O—substituted cycloalkenyl, —O—C(O)O-heteroaryl, —O—C(O)O-substitutedheteroaryl, —O—C(O)O-heterocyclic, and —O—C(O)O-substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

“Cyano” or “nitrile” refers to the group —CN.

“Cycloalkyl” refers to cyclic alkyl groups of from 3 to 10 carbon atomshaving single or multiple cyclic rings including fused, bridged, andspiro ring systems. Examples of suitable cycloalkyl groups include, forinstance, adamantyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyland the like. Such cycloalkyl groups include, by way of example, singlering structures such as cyclopropyl, cyclobutyl, cyclopentyl,cyclooctyl, and the like, or multiple ring structures such asadamantanyl, and the like.

The term “substituted cycloalkyl” refers to cycloalkyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkyl, substituted alkyl, alkoxy, substituted alkoxy, cycloalkyl,substituted cycloalkyl, cycloalkenyl, substituted cycloalkenyl, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, oxo, thioketo, carboxyl,carboxylalkyl, thioaryloxy, thioheteroaryloxy, thioheterocyclooxy,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, heteroaryl,heteroaryloxy, heterocyclyl, heterocyclooxy, hydroxyamino, alkoxyamino,nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkenyl” refers to non-aromatic cyclic alkyl groups of from 3 to10 carbon atoms having single or multiple rings and having at least onedouble bond and preferably from 1 to 2 double bonds.

The term “substituted cycloalkenyl” refers to cycloalkenyl groups havingfrom 1 to 5 substituents, or from 1 to 3 substituents, selected fromalkoxy, substituted alkoxy, cycloalkyl, substituted cycloalkyl,cycloalkenyl, substituted cycloalkenyl, acyl, acylamino, acyloxy, amino,substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano,halogen, hydroxyl, keto, thioketo, carboxyl, carboxylalkyl, thioaryloxy,thioheteroaryloxy, thioheterocyclooxy, thiol, thioalkoxy, substitutedthioalkoxy, aryl, aryloxy, heteroaryl, heteroaryloxy, heterocyclyl,heterocyclooxy, hydroxyamino, alkoxyamino, nitro, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl.

“Cycloalkynyl” refers to non-aromatic cycloalkyl groups of from 5 to 10carbon atoms having single or multiple rings and having at least onetriple bond.

“Cycloalkoxy” refers to —O-cycloalkyl.

“Cycloalkenyloxy” refers to —O-cycloalkenyl.

“Halo” or “halogen” refers to fluoro, chloro, bromo, and iodo.

“Hydroxy” or “hydroxyl” refers to the group —OH.

“Heteroaryl” refers to an aromatic group of from 1 to 15 carbon atoms,such as from 1 to 10 carbon atoms and 1 to 10 heteroatoms selected fromthe group consisting of oxygen, nitrogen, and sulfur within the ring.Such heteroaryl groups can have a single ring (such as, pyridinyl,imidazolyl or furyl) or multiple condensed rings in a ring system (forexample as in groups such as, indolizinyl, quinolinyl, benzofuran,benzimidazolyl or benzothienyl), wherein at least one ring within thering system is aromatic and at least one ring within the ring system isaromatic, provided that the point of attachment is through an atom of anaromatic ring. In certain embodiments, the nitrogen and/or sulfur ringatom(s) of the heteroaryl group are optionally oxidized to provide forthe N-oxide (N→O), sulfinyl, or sulfonyl moieties. This term includes,by way of example, pyridinyl, pyrrolyl, indolyl, thiophenyl, andfuranyl. Unless otherwise constrained by the definition for theheteroaryl substituent, such heteroaryl groups can be optionallysubstituted with 1 to 5 substituents, or from 1 to 3 substituents,selected from acyloxy, hydroxy, thiol, acyl, alkyl, alkoxy, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, substituted alkyl, substitutedalkoxy, substituted alkenyl, substituted alkynyl, substitutedcycloalkyl, substituted cycloalkenyl, amino, substituted amino,aminoacyl, acylamino, alkaryl, aryl, aryloxy, azido, carboxyl,carboxylalkyl, cyano, halogen, nitro, heteroaryl, heteroaryloxy,heterocyclyl, heterocyclooxy, aminoacyloxy, oxyacylamino, thioalkoxy,substituted thioalkoxy, thioaryloxy, thioheteroaryloxy, —SO-alkyl,—SO-substituted alkyl, —SO-aryl, —SO— heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl, andtrihalomethyl.

The term “heteroaralkyl” refers to the groups -alkylene-heteroaryl wherealkylene and heteroaryl are defined herein. This term includes, by wayof example, pyridylmethyl, pyridylethyl, indolylmethyl, and the like.

“Heteroaryloxy” refers to —O-heteroaryl.

“Heterocycle,” “heterocyclic,” “heterocycloalkyl,” and “heterocyclyl”refer to a saturated or unsaturated group having a single ring ormultiple condensed rings, including fused bridged and spiro ringsystems, and having from 3 to 20 ring atoms, including 1 to 10 heteroatoms. These ring atoms are selected from the group consisting ofnitrogen, sulfur, or oxygen, wherein, in fused ring systems, one or moreof the rings can be cycloalkyl, aryl, or heteroaryl, provided that thepoint of attachment is through the non-aromatic ring. In certainembodiments, the nitrogen and/or sulfur atom(s) of the heterocyclicgroup are optionally oxidized to provide for the N-oxide, —S(O)—, or—SO₂— moieties.

Examples of heterocycles and heteroaryls include, but are not limitedto, azetidine, pyrrole, imidazole, pyrazole, pyridine, pyrazine,pyrimidine, pyridazine, indolizine, isoindole, indole, dihydroindole,indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine,imidazolidine, imidazoline, piperidine, piperazine, indoline,phthalimide, 1,2,3,4-tetrahydroisoquinoline,4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene,benzo[b]thiophene, morpholinyl, thiomorpholinyl (also referred to asthiamorpholinyl), 1,1-dioxothiomorpholinyl, piperidinyl, pyrrolidine,tetrahydrofuranyl, and the like.

Unless otherwise constrained by the definition for the heterocyclicsubstituent, such heterocyclic groups can be optionally substituted with1 to 5, or from 1 to 3 substituents, selected from alkoxy, substitutedalkoxy, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, acyl, acylamino, acyloxy, amino, substituted amino,aminoacyl, aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl,oxo, thioketo, carboxyl, carboxylalkyl, thioaryloxy, thioheteroaryloxy,thioheterocyclooxy, thiol, thioalkoxy, substituted thioalkoxy, aryl,aryloxy, heteroaryl, heteroaryloxy, heterocyclyl, heterocyclooxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl,—SO-aryl, —SO— heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl,—SO₂-aryl, —SO₂-heteroaryl, and fused heterocycle.

“Heterocyclyloxy” refers to the group —O-heterocyclyl.

The term “heterocyclylthio” refers to the group heterocyclic-S—.

The term “heterocyclene” refers to the diradical group formed from aheterocycle, as defined herein.

The term “hydroxyamino” refers to the group —NHOH.

“Nitro” refers to the group —NO₂.

“Oxo” refers to the atom (═O).

“Sulfonyl” refers to the group SO₂-alkyl, SO₂-substituted alkyl,SO₂-alkenyl, SO₂-substituted alkenyl, SO₂-cycloalkyl, SO₂-substitutedcycloalkyl, SO₂-cycloalkenyl, SO₂-substituted cylcoalkenyl, SO₂-aryl,SO₂-substituted aryl, SO₂-heteroaryl, SO₂-substituted heteroaryl,SO₂-heterocyclic, and SO₂-substituted heterocyclic, wherein alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, cycloalkyl, substituted cycloalkyl, cycloalkenyl, substitutedcycloalkenyl, aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclic, and substituted heterocyclic are as definedherein. Sulfonyl includes, by way of example, methyl-SO₂—, phenyl-SO₂—,and 4-methylphenyl-SO₂—.

“Sulfonyloxy” refers to the group —OSO₂-alkyl, OSO₂-substituted alkyl,OSO₂-alkenyl, OSO₂-substituted alkenyl, OSO₂-cycloalkyl,OSO₂-substituted cycloalkyl, OSO₂-cycloalkenyl, OSO₂-substitutedcylcoalkenyl, OSO₂-aryl, OSO₂-substituted aryl, OSO₂-heteroaryl,OSO₂-substituted heteroaryl, OSO₂-heterocyclic, and OSO₂ substitutedheterocyclic, wherein alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, cycloalkyl, substitutedcycloalkyl, cycloalkenyl, substituted cycloalkenyl, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, heterocyclic, and substitutedheterocyclic are as defined herein.

The term “aminocarbonyloxy” refers to the group —OC(O)NRR where each Ris independently hydrogen, alkyl, substituted alkyl, aryl, heteroaryl,or heterocyclic wherein alkyl, substituted alkyl, aryl, heteroaryl andheterocyclic are as defined herein.

“Thiol” refers to the group —SH.

“Thioxo” or the term “thioketo” refers to the atom (═S).

“Alkylthio” or the term “thioalkoxy” refers to the group —S-alkyl,wherein alkyl is as defined herein. In certain embodiments, sulfur maybe oxidized to —S(O)—. The sulfoxide may exist as one or morestereoisomers.

The term “substituted thioalkoxy” refers to the group —S-substitutedalkyl.

The term “thioaryloxy” refers to the group aryl-S— wherein the arylgroup is as defined herein including optionally substituted aryl groupsalso defined herein.

The term “thioheteroaryloxy” refers to the group heteroaryl-S— whereinthe heteroaryl group is as defined herein including optionallysubstituted aryl groups as also defined herein.

The term “thioheterocyclooxy” refers to the group heterocyclyl-S—wherein the heterocyclyl group is as defined herein including optionallysubstituted heterocyclyl groups as also defined herein.

In addition to the disclosure herein, the term “substituted,” when usedto modify a specified group or radical, can also mean that one or morehydrogen atoms of the specified group or radical are each, independentlyof one another, replaced with the same or different substituent groupsas defined below.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for substituting for one or more hydrogens(any two hydrogens on a single carbon can be replaced with ═O, ═NR⁷⁰,═N—OR⁷⁰, ═N₂ or ═S) on saturated carbon atoms in the specified group orradical are, unless otherwise specified, —R⁶⁰, halo, ═O, —OR⁷⁰, —SR⁷⁰,—NR⁸⁰R⁸⁰, trihalomethyl, —CN, —OCN, —SCN, —NO, —NO₂, ═N₂, —N₃, —SO₂R⁷⁰,—SO₂O⁻M⁺, —SO₂OR⁷⁰, —OSO₂R⁷⁰, —OSO₂O⁻M⁺, —OSO₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂,—P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰,—C(O)O⁻M⁺, —C(O)OR⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰,—OC(O)R⁷⁰, —OC(S)R⁷⁰, —OC(O) O⁻M⁺, —OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰,—NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰ isselected from the group consisting of optionally substituted alkyl,cycloalkyl, heteroalkyl, heterocycloalkylalkyl, cycloalkylalkyl, aryl,arylalkyl, heteroaryl and heteroarylalkyl, each R⁷⁰ is independentlyhydrogen or R⁶⁰; each R⁸⁰ is independently R⁷⁰ or alternatively, twoR⁸⁰'s, taken together with the nitrogen atom to which they are bonded,form a 5-, 6- or 7-membered heterocycloalkyl which may optionallyinclude from 1 to 4 of the same or different additional heteroatomsselected from the group consisting of O, N and S, of which N may have —Hor C₁-C₃ alkyl substitution; and each M⁺ is a counter ion with a netsingle positive charge. Each M⁺ may independently be, for example, analkali ion, such as K⁺, Na⁺, Li⁺; an ammonium ion, such as ⁺N(R⁶⁰)₄; oran alkaline earth ion, such as [Ca^(2+]) _(0.5), [Mg²⁺]_(0.5), or[Ba²⁺]_(0.5) (“subscript 0.5 means that one of the counter ions for suchdivalent alkali earth ions can be an ionized form of a compound of theinvention and the other a typical counter ion such as chloride, or twoionized compounds disclosed herein can serve as counter ions for suchdivalent alkali earth ions, or a doubly ionized compound of theinvention can serve as the counter ion for such divalent alkali earthions). As specific examples, —NR⁸⁰R⁸⁰ is meant to include —NH₂,—NH-alkyl, N-pyrrolidinyl, N-piperazinyl, 4N-methyl-piperazin-1-yl andN-morpholinyl.

In addition to the disclosure herein, substituent groups for hydrogenson unsaturated carbon atoms in “substituted” alkene, alkyne, aryl andheteroaryl groups are, unless otherwise specified, —R⁶⁰, halo, —O⁻M⁺,—OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰, trihalomethyl, —CF₃, —CN, —OCN, —SCN,—NO, —NO₂, —N₃, —SO₂R⁷⁰, —SO₃ ⁻M⁺, —SO₃R⁷⁰, —OSO₂R⁷⁰, —OSO₃ ⁻M⁺,—OSO₃R⁷⁰, —PO₃ ⁻²(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺, —P(O)(OR⁷⁰)₂, —C(O)R⁷⁰,—C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —CO₂ ⁻M⁺, —CO₂R⁷⁰, —C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰,—C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰, —OCO₂ ⁻M⁺, —OCO₂R⁷⁰, —OC(S)OR⁷⁰,—NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰CO₂ ⁻M⁺, —NR⁷⁰CO₂R⁷⁰, —NR⁷⁰C(S)OR⁷⁰,—NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and —NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰,R⁷⁰, R⁸⁰ and M⁺are as previously defined, provided that in case ofsubstituted alkene or alkyne, the substituents are not —O⁻M⁺, —OR⁷⁰,—SR⁷⁰, or —S⁻M⁺.

In addition to the groups disclosed with respect to the individual termsherein, substituent groups for hydrogens on nitrogen atoms in“substituted” heteroalkyl and cycloheteroalkyl groups are, unlessotherwise specified, —R⁶⁰, —O⁻M⁺, —OR⁷⁰, —SR⁷⁰, —S⁻M⁺, —NR⁸⁰R⁸⁰,trihalomethyl, —CF₃, —CN, —NO, —NO₂, —S(O)₂R⁷⁰, —S(O)₂O⁻M⁺, —S(O)₂OR⁷⁰,—OS(O)₂R⁷⁰, —OS(O)₂O⁻M⁺, —OS(O)₂OR⁷⁰, —P(O)(O⁻)₂(M⁺)₂, —P(O)(OR⁷⁰)O⁻M⁺,—P(O)(OR⁷⁰)(OR⁷⁰), —C(O)R⁷⁰, —C(S)R⁷⁰, —C(NR⁷⁰)R⁷⁰, —C(O)OR⁷⁰,—C(S)OR⁷⁰, —C(O)NR⁸⁰R⁸⁰, —C(NR⁷⁰)NR⁸⁰R⁸⁰, —OC(O)R⁷⁰, —OC(S)R⁷⁰,—OC(O)OR⁷⁰, —OC(S)OR⁷⁰, —NR⁷⁰C(O)R⁷⁰, —NR⁷⁰C(S)R⁷⁰, —NR⁷⁰C(O)OR⁷⁰,—NR⁷⁰C(S)OR⁷⁰, —NR⁷⁰C(O)NR⁸⁰R⁸⁰, —NR⁷⁰C(NR⁷⁰)R⁷⁰ and—NR⁷⁰C(NR⁷⁰)NR⁸⁰R⁸⁰, where R⁶⁰, R⁷⁰, R⁸⁰ and M⁺ are as previouslydefined.

In addition to the disclosure herein, in a certain embodiment, a groupthat is substituted has 1, 2, 3, or 4 substituents, 1, 2, or 3substituents, 1 or 2 substituents, or 1 substituent.

It is understood that in all substituted groups defined above, polymersarrived at by defining substituents with further substituents tothemselves (e.g., substituted aryl having a substituted aryl group as asubstituent which is itself substituted with a substituted aryl group,which is further substituted by a substituted aryl group, etc.) are notintended for inclusion herein. In such cases, the maximum number of suchsubstitutions is three. For example, serial substitutions of substitutedaryl groups specifically contemplated herein are limited to substitutedaryl-(substituted aryl)-substituted aryl.

Unless indicated otherwise, the nomenclature of substituents that arenot explicitly defined herein are arrived at by naming the terminalportion of the functionality followed by the adjacent functionalitytoward the point of attachment. For example, the substituent“arylalkyloxycarbonyl” refers to the group (aryl)-(alkyl)-O—C(O)—.

As to any of the groups disclosed herein which contain one or moresubstituents, it is understood, of course, that such groups do notcontain any substitution or substitution patterns which are stericallyimpractical and/or synthetically non-feasible. In addition, the subjectcompounds include all stereochemical isomers arising from thesubstitution of these compounds.

The term “pharmaceutically acceptable salt” means a salt which isacceptable for administration to a patient, such as a mammal (salts withcounterions having acceptable mammalian safety for a given dosageregime). Such salts can be derived from pharmaceutically acceptableinorganic or organic bases and from pharmaceutically acceptableinorganic or organic acids. “Pharmaceutically acceptable salt” refers topharmaceutically acceptable salts of a compound, which salts are derivedfrom a variety of organic and inorganic counter ions well known in theart and include, by way of example only, sodium, potassium, calcium,magnesium, ammonium, tetraalkylammonium, and the like; and when themolecule contains a basic functionality, salts of organic or inorganicacids, such as hydrochloride, hydrobromide, formate, tartrate, besylate,mesylate, acetate, maleate, oxalate, and the like.

The term “salt thereof” means a compound formed when a proton of an acidis replaced by a cation, such as a metal cation or an organic cation andthe like. Where applicable, the salt is a pharmaceutically acceptablesalt, although this is not required for salts of intermediate compoundsthat are not intended for administration to a patient. By way ofexample, salts of the present compounds include those wherein thecompound is protonated by an inorganic or organic acid to form a cation,with the conjugate base of the inorganic or organic acid as the anioniccomponent of the salt.

“Solvate” refers to a complex formed by combination of solvent moleculeswith molecules or ions of the solute. The solvent can be an organiccompound, an inorganic compound, or a mixture of both. Some examples ofsolvents include, but are not limited to, methanol,N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, and water.When the solvent is water, the solvate formed is a hydrate.

“Stereoisomer” and “stereoisomers” refer to compounds that have sameatomic connectivity but different atomic arrangement in space.Stereoisomers include cis-trans isomers, E and Z isomers, enantiomers,and diastereomers.

“Tautomer” refers to alternate forms of a molecule that differ only inelectronic bonding of atoms and/or in the position of a proton, such asenol-keto and imine-enamine tautomers, or the tautomeric forms ofheteroaryl groups containing a —N═C(H)—NH— ring atom arrangement, suchas pyrazoles, imidazoles, benzimidazoles, triazoles, and tetrazoles. Aperson of ordinary skill in the art would recognize that othertautomeric ring atom arrangements are possible.

It will be appreciated that the term “or a salt or solvate orstereoisomer thereof” is intended to include all permutations of salts,solvates and stereoisomers, such as a solvate of a pharmaceuticallyacceptable salt of a stereoisomer of subject compound.

“Pharmaceutically effective amount” and “therapeutically effectiveamount” refer to an amount of a compound sufficient to treat a specifieddisorder or disease or one or more of its symptoms and/or to prevent theoccurrence of the disease or disorder. In reference to tumorigenicproliferative disorders, a pharmaceutically or therapeutically effectiveamount comprises an amount sufficient to, among other things, cause thetumor to shrink or decrease the growth rate of the tumor.

“Patient” refers to human and non-human subjects, especially mammaliansubjects.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition in a patient, such as amammal (particularly a human) that includes: (a) preventing the diseaseor medical condition from occurring, such as, prophylactic treatment ofa subject; (b) ameliorating the disease or medical condition, such as,eliminating or causing regression of the disease or medical condition ina patient; (c) suppressing the disease or medical condition, for exampleby, slowing or arresting the development of the disease or medicalcondition in a patient; or (d) alleviating a symptom of the disease ormedical condition in a patient.

Representative Embodiments

The following substituents and values are intended to providerepresentative examples of various aspects and embodiments. Theserepresentative values are intended to further define and illustrate suchaspects and embodiments and are not intended to exclude otherembodiments or to limit the scope of this invention. In this regard, therepresentation that a particular value or substituent is preferred isnot intended in any way to exclude other values or substituents fromthis invention unless specifically indicated.

These compounds may contain one or more chiral centers and therefore,the embodiments are directed to racemic mixtures; pure stereoisomers(i.e., enantiomers or diastereomers); stereoisomer-enriched mixtures andthe like unless otherwise indicated. When a particular stereoisomer isshown or named herein, it will be understood by those skilled in the artthat minor amounts of other stereoisomers may be present in thecompositions unless otherwise indicated, provided that the desiredutility of the composition as a whole is not eliminated by the presenceof such other isomers.

The compositions of the present disclosure include compounds of formulaeI-V, shown below. Pharmaceutical compositions and methods of the presentdisclosure also contemplate compounds of formulae I-V.

Formula I

In one of its composition aspects, the present embodiments provide acompound of formula (I):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

G is halogen or —NY²Ar¹;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl;

or a salt or stereoisomer thereof.

In formula (I), G is halogen or —NY²Ar¹. In certain embodiments, G ishalogen. In certain embodiments, G is —NY²Ar¹.

In formula (I), Ar¹ is selected from aryl, substituted aryl, heteroaryl,and substituted heteroaryl. In certain embodiments, Ar¹ is aryl orsubstituted aryl. In certain embodiments, Ar¹ is aryl. In certainembodiments, Ar¹ is substituted aryl. In certain embodiments, Ar¹ isheteroaryl or substituted heteroaryl. In certain embodiments, Ar¹ isheteroaryl. In certain embodiments, Ar¹ is substituted heteroaryl.

In certain embodiments, a compound of formula (I) is of the formula:

wherein

H¹ and H² are hydrogen.

In certain embodiments, H¹ and H² are hydrogen with cis relativeconfiguration. In certain embodiments, H¹ and H² are hydrogen with transrelative configuration.

Formula II

In one of its composition aspects, the present embodiments provide acompound of formula (II):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IIa):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and

H¹ and H² are hydrogen with cis relative configuration;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IIb):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl; and

H¹ and H² are hydrogen with trans relative configuration.

or a salt or stereoisomer thereof.

Reference to formula (II) is meant to include compounds of formula (II)and (IIa)-(IIb).

In formulae (II), Ar¹ is selected from aryl, substituted aryl,heteroaryl, and substituted heteroaryl. In certain embodiments, Ar¹ isaryl or substituted aryl. In certain embodiments, Ar¹ is aryl. Incertain embodiments, Ar¹ is substituted aryl. In certain embodiments,Ar¹ is heteroaryl or substituted heteroaryl. In certain embodiments, Ar¹is heteroaryl. In certain embodiments, Ar¹ is substituted heteroaryl.

In formula (IIa) and (IIb), in certain embodiments, the compound isoptically active. In certain embodiments, there is an enantiomericexcess of 90% or more. In certain embodiments, there is an enantiomericexcess of 95% or more. In certain embodiments, there is an enantiomericexcess of 99% or more.

Formula III

In one of its composition aspects, the present embodiments provide acompound of formula (III):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

Q is C or N; and

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, halogen, cyano, hydroxyl, alkoxy, substitutedalkoxy, amino, substituted amino, acylamino, aminocarbonylamino,alkoxycarbonylamino, acyl, carboxyl, carboxyl ester, aminoacyl,aminocarbonyloxy, nitro, sulfonyl, sulfonylamino, aminosulfonyl, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IIa):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

Q is C or N;

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, halogen, cyano, hydroxyl, alkoxy, substitutedalkoxy, amino, substituted amino, acylamino, aminocarbonylamino,alkoxycarbonylamino, acyl, carboxyl, carboxyl ester, aminoacyl,aminocarbonyloxy, nitro, sulfonyl, sulfonylamino, aminosulfonyl, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl; and

H¹ and H² are hydrogen with cis relative configuration;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IIIb):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

Q is C or N;

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, halogen, cyano, hydroxyl, alkoxy, substitutedalkoxy, amino, substituted amino, acylamino, aminocarbonylamino,alkoxycarbonylamino, acyl, carboxyl, carboxyl ester, aminoacyl,aminocarbonyloxy, nitro, sulfonyl, sulfonylamino, aminosulfonyl, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl; and

H¹ and H² are hydrogen with trans relative configuration;

or a salt or stereoisomer thereof.

Reference to formula (III) is meant to include compounds of formula(III) and (IIIa)-(IIIb).

In formula (III), Q is C or N. In certain embodiments, Q is C. Incertain embodiments, Q is N.

In formula (IIIa) and (IIIb), in certain embodiments, the compound isoptically active. In certain embodiments, there is an enantiomericexcess of 90% or more. In certain embodiments, there is an enantiomericexcess of 95% or more. In certain embodiments, there is an enantiomericexcess of 99% or more.

Formula IV

In one of its composition aspects, the present embodiments provide acompound of formula (IV):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, halogen, cyano, hydroxyl, alkoxy, substitutedalkoxy, amino, substituted amino, acylamino, aminocarbonylamino,alkoxycarbonylamino, acyl, carboxyl, carboxyl ester, aminoacyl,aminocarbonyloxy, nitro, sulfonyl, sulfonylamino, aminosulfonyl, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IVa):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, halogen, cyano, hydroxyl, alkoxy, substitutedalkoxy, amino, substituted amino, acylamino, aminocarbonylamino,alkoxycarbonylamino, acyl, carboxyl, carboxyl ester, aminoacyl,aminocarbonyloxy, nitro, sulfonyl, sulfonylamino, aminosulfonyl, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl; and

H¹ and H² are hydrogen with cis relative configuration;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IVb):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl,substituted alkynyl, halogen, cyano, hydroxyl, alkoxy, substitutedalkoxy, amino, substituted amino, acylamino, aminocarbonylamino,alkoxycarbonylamino, acyl, carboxyl, carboxyl ester, aminoacyl,aminocarbonyloxy, nitro, sulfonyl, sulfonylamino, aminosulfonyl, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl; and

H¹ and H² are hydrogen with trans relative configuration;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (IVc):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

R¹² is selected from hydrogen and halogen;

R¹³ and R¹⁶ are hydrogen;

R¹⁴ is an alkoxy substituted with hydroxyl and optionally substitutedwith one or more alkyl groups;

R¹⁵ is substituted heterocyclyl;

or a salt or stereoisomer thereof.

In some embodiments of the present compounds, such as compounds offormula (IVc), R¹⁴ has the formula —O-Alk-OR²⁵, where Alk is anoptionally substituted alkylene group and R²⁵ is selected from H and anoptionally substituted alkyl group. In some embodiments, Alk is analkylene group. In some embodiments, Alk is a substituted alkylenegroup. In some embodiments of such compounds, Alk represents anoptionally substituted ethylene or propylene moiety. In certainembodiments, Alk is an ethylene moiety. In certain embodiments, Alk is apropylene moiety. In certain embodiments, Alk is a substituted ethylenemoiety. In certain embodiments, Alk is a substituted propylene moiety.In some embodiments, Alk is substituted with one, two or three C₁₋₆alkyl groups, such as, by way of example, one or two methyl groupssubstituted on the same or different carbon atoms of the alkylene chain.

Reference to formula (IV) is meant to include compounds of formula (IV)and (IVa)-(IVc).

In formula (IVa) and (IVc), in certain embodiments, the compound isoptically active. In certain embodiments, there is an enantiomericexcess of 90% or more. In certain embodiments, there is an enantiomericexcess of 95% or more. In certain embodiments, there is an enantiomericexcess of 99% or more.

Formula V

In one of its composition aspects, the present embodiments provide acompound of formula (V):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

R¹², R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogen, cyano, hydroxyl, alkoxy, substituted alkoxy, amino,substituted amino, acylamino, aminocarbonylamino, alkoxycarbonylamino,acyl, carboxyl, carboxyl ester, aminoacyl, aminocarbonyloxy, nitro,sulfonyl, sulfonylamino, aminosulfonyl, sulfur pentafluoride, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl, substituted heterocyclyl,heterocyclyloxy, and substituted heterocyclyloxy, or R¹⁴ and R¹⁵together with the carbon atoms to which they are attached form aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, orsubstituted heterocyclyl;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (Va):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

R¹², R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogen, cyano, hydroxyl, alkoxy, substituted alkoxy, amino,substituted amino, acylamino, aminocarbonylamino, alkoxycarbonylamino,acyl, carboxyl, carboxyl ester, aminoacyl, aminocarbonyloxy, nitro,sulfonyl, sulfonylamino, aminosulfonyl, sulfur pentafluoride, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl, substituted heterocyclyl,heterocyclyloxy, and substituted heterocyclyloxy, or R¹⁴ and R¹⁵together with the carbon atoms to which they are attached form aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, orsubstituted heterocyclyl; and

H¹ and H² are hydrogen with cis relative configuration;

or a salt or stereoisomer thereof.

In one of its composition aspects, the present embodiments provide acompound of formula (Vb):

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

Y¹ and Y² are independently selected from hydrogen and alkyl;

R¹², R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, halogen, cyano, hydroxyl, alkoxy, substituted alkoxy, amino,substituted amino, acylamino, aminocarbonylamino, alkoxycarbonylamino,acyl, carboxyl, carboxyl ester, aminoacyl, aminocarbonyloxy, nitro,sulfonyl, sulfonylamino, aminosulfonyl, sulfur pentafluoride, aryl,substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl,substituted heteroaryl, heterocyclyl, substituted heterocyclyl,heterocyclyloxy, and substituted heterocyclyloxy, or R¹⁴ and R¹⁵together with the carbon atoms to which they are attached form aryl,substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl, orsubstituted heterocyclyl; and

H¹ and H² are hydrogen with trans relative configuration;

or a salt or stereoisomer thereof.

Reference to formula (V) is meant to include compounds of formula (V)and (Va)-(Vb)

In formula (Va) and (Vb), in certain embodiments, the compound isoptically active. In certain embodiments, there is an enantiomericexcess of 90% or more. In certain embodiments, there is an enantiomericexcess of 95% or more. In certain embodiments, there is an enantiomericexcess of 99% or more.

Certain Embodiments of Formulae I-V

With reference to formulae I-V, the formula

includes at least two chiral centers, and thus at least fourstereoisomers. For clarity the numbering of the ring system is shownbelow with optional substituents omitted.

With continued reference to formulae I-V, (7,8a) cis diastereomer hasthe structure:

and the (7,8a) trans diastereomer:

In formulae I-V, R¹, R², R³, R⁴, R⁵, and R⁶ are independently selectedfrom hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl,aminoacyloxy, oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl,carboxylalkyl, thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy,hydroxyamino, alkoxyamino, nitro, —SO-alkyl, —SO— substituted alkyl,—SO-aryl, —SO-hetero aryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryland —SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ andR⁴ together form an oxo group; or R⁵ and R⁶ together form an oxo group.

In certain embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are independentlyselected from hydrogen, alkyl, substituted alkyl, cyano, halogen,hydroxyl, acyl, aminoacyl, and nitro; or R¹ and R² together form an oxogroup; or R³ and R⁴ together form an oxo group; or R⁵ and R⁶ togetherform an oxo group.

In certain embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are independentlyselected from hydrogen, alkyl, substituted alkyl, halogen, and hydroxyl;or R¹ and R² together form an oxo group; or R³ and R⁴ together form anoxo group; or R⁵ and R⁶ together form an oxo group.

In certain embodiments, R¹ and R² are each hydrogen. In certainembodiments, R¹ and R² are each alkyl, such as C₁-C₆ alkyl, or C₁-C₃alkyl. In certain embodiments, R¹ and R² are each methyl.

In certain embodiments, at least one of R³ and R⁴ is hydrogen. Incertain embodiments, R³ and R⁴ are each hydrogen. In certainembodiments, at least one of R³ and R⁴ is halogen or hydroxyl. Incertain embodiments, at least one of R³ and R⁴ is halogen. In certainembodiments, one of R³ and R⁴ is halogen. In certain embodiments, atleast one of R³ and R⁴ is F. In certain embodiments, one of R³ and R⁴ isF. In certain embodiments, R³ and R⁴ are each halogen. In certainembodiments, R³ and R⁴ are each F. In certain embodiments, at least oneof R³ and R⁴ is hydroxyl. In certain embodiments, one of R³ and R⁴ ishydroxyl.

In certain embodiments, at least one of R⁵ and R⁶ is hydrogen. Incertain embodiments, R⁵ and R⁶ are each hydrogen.

In certain embodiments, R¹ and R² together form an oxo group; or R³ andR⁴ together form an oxo group; or R⁵ and R⁶ together form an oxo group.In certain embodiments,

R¹ and R² together form an oxo group. In certain embodiments, R³ and R⁴together form an oxo group. In certain embodiments, R⁵ and R⁶ togetherform an oxo group.

In certain embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are each hydrogen.

In certain embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are independentlyselected from hydrogen, alkyl, and substituted alkyl. In certainembodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected fromhydrogen, alkoxy, substituted alkoxy, aryloxy, hydroxyamino,alkoxyamino, and nitro. In certain embodiments, R¹, R², R³, R⁴, R⁵, andR⁶ are independently selected from hydrogen, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, andazido. In certain embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ areindependently selected from hydrogen, cyano, halogen, and hydroxyl. Incertain embodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are independentlyselected from hydrogen, carboxyl, carboxylalkyl, thiol, thioalkoxy, andsubstituted thioalkoxy. In certain embodiments, R¹, R², R³, R⁴, R⁵, andR⁶ are independently selected from hydrogen and aryl. In certainembodiments, R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected fromhydrogen, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Incertain embodiments, R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group.

In formulae I-V, R⁷, R⁸, R⁹, and R¹⁰ are independently selected fromhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R⁷ and R⁸ together form an oxo group; or R⁹ and R¹⁰together form an oxo group.

In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are independently selectedfrom hydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy,cyano, halogen, acyl, aminoacyl, and nitro; or R⁷ and R⁸ together forman oxo group. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ areindependently selected from hydrogen, alkyl, substituted alkyl, andhalogen; or R⁷ and R⁸ together form an oxo group.

In certain embodiments, R⁷ and R⁸ are each hydrogen. In certainembodiments, R⁷ and R⁸ are each alkyl, such as C₁-C₆ alkyl, or C₁-C₃alkyl. In certain embodiments, R⁷ and R⁸ are each methyl. In certainembodiments, R⁷ and R⁸ together form an oxo group.

In certain embodiments, at least one of R⁹ and R¹⁰ is hydrogen. Incertain embodiments, R⁹ and R¹⁰ are each hydrogen. In certainembodiments, at least one of R⁹ and R¹⁰ is alkyl, such as C₁-C₆ alkyl,or C₁-C₃ alkyl. In certain embodiments, at least one of R⁹ and R¹⁰ ismethyl. In certain embodiments, one of R⁹ and R¹⁰ is methyl. In certainembodiments, R⁹ and R¹⁰ together form an oxo group.

In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are independently selectedfrom hydrogen, alkyl, and substituted alkyl. In certain embodiments, R⁷,R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkoxy,substituted alkoxy, aryloxy, hydroxyamino, alkoxyamino, and nitro. Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are independently selected fromhydrogen, acyl, acylamino, acyloxy, amino, substituted amino, aminoacyl,aminoacyloxy, oxyaminoacyl, and azido. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are independently selected from hydrogen, cyano, halogen,and hydroxyl. In certain embodiments, R⁷, R⁸, R⁹, and R¹⁰ areindependently selected from hydrogen, carboxyl, carboxylalkyl, thiol,thioalkoxy, and substituted thioalkoxy. In certain embodiments, R⁷, R⁸,R⁹, and R¹⁰ are independently selected from hydrogen and aryl. Incertain embodiments, R⁷, R⁸, R⁹, and R¹⁰ are independently selected fromhydrogen, —SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl,—SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl. Incertain embodiments, R⁷ and R⁸ together form an oxo group; or R⁹ and R¹⁰together form an oxo group.

In formulae I-V, R¹¹ is selected from alkyl, substituted alkyl, hydroxy,alkoxy, substituted alkoxy, amino, substituted amino, carboxyl, carboxylester, cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substitutedalkenyl, alkynyl, and substituted alkynyl.

In certain embodiments, R¹¹ is selected from alkoxy, substituted alkoxy,cyano, halogen, acyl, aminoacyl, and nitro. In certain embodiments, R¹¹is cyano, halogen, acyl, aminoacyl, or nitro. In certain embodiments,R¹¹ is fluoro or cyano. In certain embodiments, R¹¹ is fluoro. Incertain embodiments, R¹¹ is cyano. In certain embodiments, R¹¹ is acylor aminoacyl. In certain embodiments, R¹¹ is nitro.

In formulae I-V, R²⁰ is selected from hydrogen, alkyl, and substitutedalkyl. In certain embodiments, R²⁰ is hydrogen. In certain embodiments,R²⁰ is alkyl. In certain embodiments, R²⁰ is substituted alkyl.

In formulae I-V, Y¹ and Y² are independently selected from hydrogen andalkyl. In certain embodiments, Y¹ and Y² are hydrogen. In certainembodiments, Y¹ and Y² are alkyl.

In formulae III-V, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, halogen, cyano, hydroxyl, alkoxy,substituted alkoxy, amino, substituted amino, acylamino,aminocarbonylamino, alkoxycarbonylamino, acyl, carboxyl, carboxyl ester,aminoacyl, aminocarbonyloxy, nitro, sulfonyl, sulfonylamino,aminosulfonyl, sulfur pentafluoride, aryl, substituted aryl, cycloalkyl,substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclyl, substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from hydrogen, alkyl, substituted alkyl, halogen, cyano,hydroxyl, alkoxy, substituted alkoxy, amino, substituted amino,acylamino, aminocarbonylamino, acyl, aminoacyl, aminocarbonyloxy, sulfurpentafluoride, aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from hydrogen, alkyl, halogen, cyano, hydroxyl, alkoxy,substituted alkoxy, acylamino, aminocarbonylamino, sulfur pentafluoride,cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl,heterocyclyl, substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy, or R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form aryl, substituted aryl, heteroaryl, substitutedheteroaryl, heterocyclyl, or substituted heterocyclyl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from hydrogen, halogen, cycloalkyl, substituted cycloalkyl,heteroaryl, and substituted heteroaryl. In certain embodiments, R¹²,R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from hydrogen, alkoxy,substituted alkoxy, heteroaryl, and substituted heteroaryl. In certainembodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromhydrogen, alkyl, alkoxy, substituted alkoxy, and cyano. In certainembodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromhydrogen, alkyl, heteroaryl, and substituted heteroaryl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from hydrogen, alkyl, and substituted alkyl. In certainembodiments, at least one of R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are hydrogen.In certain embodiments, one of R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ is hydrogen.In certain embodiments, two of R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are hydrogen.In certain embodiments, three of R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ arehydrogen. In certain embodiments, R¹² and R¹⁶ are hydrogen. In certainembodiments, R¹², R¹³, and R¹⁶ are hydrogen. In certain embodiments,R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from alkyl andsubstituted alkyl. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶are independently selected from alkyl, such as C₁-C₆ alkyl, or C₁-C₃alkyl. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ areindependently selected from methyl. In certain embodiments, R¹², R¹³,R¹⁴, R¹⁵, and R¹⁶ are independently selected from alkenyl, substitutedalkenyl, alkynyl, and substituted alkynyl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from halogen, cyano, hydroxyl, alkoxy, and substituted alkoxy.In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from halogen. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, andR¹⁶ are independently selected from fluoro. In certain embodiments, R¹²,R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from cyano. In certainembodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromhydroxyl. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ areindependently selected from alkoxy. In certain embodiments, R¹², R¹³,R¹⁴, R¹⁵, and R¹⁶ are independently selected from C₁-C₆ alkoxy. Incertain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from C₁-C₃ alkoxy, such as methoxy, isopropoxy, and the like.In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from substituted alkoxy. In certain embodiments, R¹², R¹³, R¹⁴,R¹⁵, and R¹⁶ are independently selected from C₁-C₆ substituted alkoxy.In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from C₁-C₃ substituted alkoxy. In certain embodiments, thesubstituted alkoxy group is substituted with one or more groups selectedfrom halogen, hydroxyl, alkyl (e.g., C₁-C₆ alkyl, such as C₁-C₃ alkyl,including methyl).

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from amino and substituted amino. In certain embodiments, R¹²,R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected from acylamino,aminocarbonylamino, alkoxycarbonylamino, acyl, carboxyl, carboxyl ester,aminoacyl, and aminocarbonyloxy. In certain embodiments, R¹², R¹³, R¹⁴,R¹⁵, and R¹⁶ are independently selected from acylamino andaminocarbonylamino. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶are independently selected from acylamino. In certain embodiments, R¹²,R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromaminocarbonylamino.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from nitro, sulfonyl, sulfonylamino, aminosulfonyl, and sulfurpentafluoride. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ areindependently selected from sulfur pentafluoride.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from aryl, substituted aryl, cycloalkyl, substitutedcycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl,substituted heterocyclyl, heterocyclyloxy, and substitutedheterocyclyloxy. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ areindependently selected from aryl and substituted aryl. In certainembodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromcycloalkyl and substituted cycloalkyl. In certain embodiments, R¹², R¹³,R¹⁴, R¹⁵, and R¹⁶ are independently selected from cycloalkyl. Forexample, cycloalkyl groups may include, but are not limited to, C₃-C₈cycloalkyl, including C₃-C₆ cycloalkyl, such as cyclohexyl, cyclopentyl,cyclobutyl, cyclopropyl. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, andR¹⁶ are independently selected from cyclopropyl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from heteroaryl, substituted heteroaryl, heterocyclyl andsubstituted heterocyclyl. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵,and R¹⁶ are independently selected from heteroaryl. In certainembodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromsubstituted heteroaryl. In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, andR¹⁶ are independently selected from heterocyclyl. In certainembodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independently selected fromsubstituted heterocyclyl. Examples of heterocycles and heteroarylsinclude, but are not limited to, tetrazolyl and substituted tetrazolyl.For example, a tetrazolyl group may be substituted with one or moregroups, such as an oxo, alkyl, e.g., C₁-C₆ alkyl, including C₁-C₃ alkyl,such as methyl, and the like.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from heterocyclyloxy and substituted heterocyclyloxy. Incertain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from heterocyclyloxy. In certain embodiments, R¹², R¹³, R¹⁴,R¹⁵, and R¹⁶ are independently selected from substitutedheterocyclyloxy. In certain embodiments, heterocyclyloxy and substitutedheterocyclyloxy groups include a 3 to 8-membered ring, such as a 3 to6-membered ring, that includes 1 to 5 heteroatoms, or 1 to 3heteroatoms, such as 1 heteroatom (e.g., oxygen, nitrogen or sulfur).For example, heterocyclyloxy and substituted heterocyclyloxy groups mayinclude rings such as, but not limited to, tetrahydropyran,tetrahydrofuran, oxetane, piperidine, and the like. Substitutedheterocyclyloxy groups may be substituted with one or more groups, suchas alkyl, e.g., C₁-C₆ alkyl, including C₁-C₃ alkyl, such as methyl,isopropyl, and the like.

In certain embodiments, R¹⁴ and R¹⁵ together with the carbon atoms towhich they are attached form aryl, substituted aryl, heteroaryl,substituted heteroaryl, heterocyclyl, or substituted heterocyclyl. Incertain embodiments, R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form an aryl or heteroaryl 5 to 10-membered ring. Incertain embodiments, R¹⁴ and R¹⁵ together with the carbon atoms to whichthey are attached form a heterocyclyl or substituted heterocyclyl 5 to10-membered ring. For example, R¹⁴ and R¹⁵ together with the carbonatoms to which they are attached may form a tetrazolooxazinyl group,which may be substituted with one or more groups, such as alkyl, e.g.,C₁-C₆ alkyl, including C₁-C₃ alkyl, such as methyl.

In certain embodiments, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are independentlyselected from hydrogen, methyl, fluoro, hydroxyl, methoxy, isopropoxy,cyano, sulfur pentafluoride, —OCHF₂, OCH₂CH₂OH, —OCH₂CH(OH)CH₃,—OCH₂C(CH₃)₂OH, —OCH₂CH(CH₃)OH, —OC(CH₃)₂CH₂OH,

In certain embodiments, including those of formulae (I-V), R¹² isselected from hydrogen and halogen. In certain embodiments, R¹² ishydrogen. In certain embodiments, R¹² is halogen. In certainembodiments, R¹² is fluoro.

In certain embodiments, R¹³ and R¹⁶ are hydrogen.

In certain embodiments, R¹⁴ is a substituted alkoxy. In certainembodiments, R¹⁴ is a C₁-C₆ substituted alkoxy, such as a C₁-C₃substituted alkoxy. In certain embodiments, R¹⁴ is a substitutedpropoxy. In certain embodiments, R¹⁴ is a substituted ethoxy. In certainembodiments, R¹⁴ is an alkoxy substituted with hydroxyl and optionallysubstituted with one or more alkyl groups. In certain embodiments, R¹⁴is an alkoxy substituted with hydroxyl. In certain embodiments, R¹⁴ isan alkoxy substituted with hydroxyl and an alkyl group, such as a C₁-C₆alkyl group, such as a C₁-C₃ alkyl group, such as a methyl group. Incertain embodiments, R¹⁴ is an alkoxy substituted with hydroxyl and twoalkyl groups. In certain embodiments, the alkyl substituents are eachindependently a C₁-C₆ alkyl group, such as a C₁-C₃ alkyl group, such asa methyl group. In certain embodiments, R¹⁴ is an ethoxy groupsubstituted with hydroxyl. In certain embodiments, R¹⁴ is an ethoxygroup substituted with hydroxyl and an alkyl group, such as a C₁-C₆alkyl group, such as a C₁-C₃ alkyl group, such as a methyl group. Incertain embodiments, R¹⁴ is an ethoxy group substituted with hydroxyland two alkyl groups. In certain embodiments, the alkyl substituents areeach independently a C₁-C₆ alkyl group, such as a C₁-C₃ alkyl group,such as a methyl group.

In certain embodiments, R¹⁵ is an optionally substituted heterocyclyl.In certain embodiments, R¹⁵ is an optionally substituted tetrazolone ofthe formula

where R²⁶ is —H, a haloalkyl group or an alkyl group, such as a C₁-C₆alkyl group, such as a C₁-C₃ alkyl group, such as a methyl group. Insome embodiments, compounds of formula (IV), such as compounds offormula (IVc), have R¹⁵ being tetrazolone. In certain embodiments,including those of formula (IV), such as formula (IVc), R¹⁵ is atetrazolone (e.g., an optionally substituted tetrazolone as describedabove) and R¹² is a halo group, such as fluoro.

Particular compounds of interest are shown illustrated the followingtable.

TABLE 1

R¹/R² R³/R⁴ R⁵/R⁶ R⁷/R⁸ R⁹/R¹⁰ R¹¹ Y¹ Y² Ar¹ H¹/H²  1 H/H H/H H/HCH₃/CH₃ H/H F H H

mixture of diastereomers  2 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer  3 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer  4 H/H H/H H/H CH₃/CH₃ H/H F H H

Racemic (single diastereomer)  5 H/H H/H H/H CH₃/CH₃ H/H F H H

Racemic cis diastereomer  6 H/H H/H H/H CH₃/CH₃ H/H F H H

mixture of diastereomers  7 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer  8 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer  9 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 10 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 11 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 12 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 13 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 14 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 15 H/H H/H H/H CH₃/CH₃ H/H F H H

H/H 16 H/H H/H H/H CH₃/CH₃ H/H F H H

racemic 17 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 18 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7S, 8aR 19 H/H H/H H/H CH₃/CH₃ H/H F H H

trans 20 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 21 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 22 (p-toluene- sulfonic salt) H/H H/H H/H CH₃/CH₃ H/HF H H

7R, 8aS 23 (p-toluene- sulfonic salt) H/H H/H H/H CH₃/CH₃ H/H F H H

7R, 8aS 24 H/H H/H H/H H/H H/H F H H

racemic (single diastereomer) 25 CH₃/CH₃ H/H H/H H/H H/H F H H

racemic (single diastereomer) 26 CH₃/CH₃ H/H H/H H/H H/H F H H

racemic (single diastereomer) 27 H/H H/H H/H H/H H/H F H H

racemic (single diastereomer) 28 H/H H/H H/H H/H H/H F H H

racemic (single diastereomer) 29 H/H H/H H/H H/H H/H F H H

racemic (single diastereomer) 30 CH₃/CH₃ H/H H/H ═O H/H F H H

racemic (single diastereomer) 31 ═O H/H H/H CH₃/CH₃ H/H F H H

racemic (7, 8a cis) 32 ═O H/H H/H CH₃/CH₃ H/H F H H

racemic (7, 8a trans) 33 (formic salt) ═O H/H H/H CH₃/CH₃ H/H F H H

racemic (7, 8a trans) 34 CH₃/CH₃ H/H H/H H/H H/H F H H

single enantiomer 35 CH₃/CH₃ H/H H/H H/H H/H F H H

single enantiomer 36 CH₃/CH₃ H/H H/H H/H H/H F H H

single enantiomer 37 CH₃/CH₃ H/H H/H H/H H/H F H H

single enantiomer 38 H/H H/H H/H CH₃/CH₃ H/H F H H

racemic (single diastereomer) 39 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 40 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 41 H/H H/H H/H CH₃/CH₃ H/H F H H

racemic (single diastereomer) 42 H/H F/F H/H CH₃/CH₃ H/H F H H

trans (7S, 8aR) 43 H/H F/F H/H CH₃/CH₃ H/H F H H

trans (7S, 8aR) 44 H/H F/F H/H CH₃/CH₃ H/H F H H

trans 45 H/H F/F H/H CH₃/CH₃ H/H F H H

cis 46 H/H F/F H/H CH₃/CH₃ H/H F H H

cis 47 H/H H/H H/H CH₃/CH₃ H/H F H H

mixture of two diastereomers 48 H/H H/H H/H CH₃/CH₃ H/H F H H

7R, 8aS 49 H/H H/H H/H CH₃/CH₃ H/H F H H

two diastereomers 50 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 51 H/H H/H H/H CH₃/CH₃ H/H F H H

racemic 52 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 53 H/H H/H H/H CH₃/CH₃ H/H F H H

cis racemic (single diastereomer) 54 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 55 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 56 H/H H/H H/H CH₃/CH₃ H/H CN H H

cis racemic (single diastereomer) 57 H/H H/H H/H CH₃/CH₃ H/H F H H

two diastereomers (7, 8a- cis) 58 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer (7, 8a-cis) 59 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 60 H/H H/H H/H CH₃/CH₃ H/H F H H

two diastereomers (7, 8a- cis) 61 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 62 H/H H/H H/H CH₃/CH₃ H/H F H H

single enantiomer 63 H/H H/H H/H CH₃/CH₃ H/H F H H

racemic (single diastereomer) 64 H/H H/  

 F (R) H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aR 65 H/H H/  

 F (R) H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7S, 8aR 66 H/H H/H H/H CH₃/CH₃ H/H CN H H

(cis) racemic 67 H/H H/  

 F (S) H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aR 68 H/H H/  

 OH (R) H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aR 69 (p-toluene- sulfonic salt) H/H H/H H/H CH₃/CH₃ H/CH₃ F H H

racemic (single diastereomer) 70 (formic acid) H/H H/H H/H CH₃/CH₃ H/CH₃F H H

racemic (single diastereomer) 71 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 72 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 73 (formic acid) H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 74 (formic acid) H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 75 (formic acid) H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 76 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS 77 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 78 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS 79 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS 80 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS 81 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 82 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 83 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS 84 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS 85 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 86 (pamoic acid) H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 87 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 88 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 89 H/H H/H H/H CH₃/CH₃ H/H F H H

 

 H/  

 H 7R, 8aS 90 H/H H/H H/H CH₃/CH₃ H/H CN H H

 

 H/  

 H 7R, 8aS

Particular compounds of interest, and salts or solvates or stereoisomersthereof, include:

Compounds 1-4:N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-amine)pyrimidine-2,4-diamine;

Compound 5:N2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compounds 6-10:(R/S,S/R,R/R,S/S)—N²-(4-(1-isopropylpiperidin-4-yloxy)-3-(difluoromethoxy)phenyl)-5-fluoro-N⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compounds 11-14:(R/S,S/R,R/R,S/S)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-yl)N2-(3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl)pyrimidine-2,4-diamine;

Compounds 16-21:octahydro-5,5-dimethylindolizin-7ylamino)-5-fluoropyrimidin-2-yl-amino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 17:1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7ylamino)-5-fluoropyrimidin-2-yl-amino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 18:1-(5-(4-((7S,8aR)-octahydro-5,5-dimethylindolizin-7ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 22:1-(5-(4-((7R,8aS)-5,5-dimethyl-octahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-methoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 23:1-(5-(4-((7R,8aS)-5,5-dimethyl-octahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-isopropoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 24:(±)-N²-(4-cyclopropyl-2-fluro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydroindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 26:N2-(4-cyclopropyl-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-yl)phenylamino)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-2,4-diamine;

Compound 27:(±)-1-(5-(5-fluoro-4-(octahydroindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)one;

Compound 28:(±)-N²-(4-cyclopropyl-2-fluoro-5-(5-methyl-1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydroindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 30:N2-{4-cyclopropyl-6-fluoro-[3-(4-methyl)-1,2,3,4-tetrazol-5-one-1-yl]}phenyl-5-fluoro-N4(7-amino-hexahydro-3,3-dimethylindolizin-5(1H)-one))2,4-pyrimidinediamine;

Compounds 31-33:1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 31:1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 32:1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 33:1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-oneformate;

Compounds 34 and 35:1-(5-(5-fluoro-4-(octahydro-3,3-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 38:1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compounds 39-40:1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 41:5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile;

Compound 42:1-(5-(4-((7S,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 43:N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-N⁴-((7S,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 44:(4-((7S,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-methylbenzonitrile;

Compound 45:1-(5-(4-((7R,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 46:N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-N⁴-((7R,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 47:1-(2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 48:1-(2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 49:1-(2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 50:1-(2-((5)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 51:1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-oxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 52:1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 53:(±)-N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compounds 54 and 55:(±)-N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 56:(±)-2-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenylamino)-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carbonitrile;

Compound 57:2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile;

Compounds 58-59:2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile;

Compound 60:2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile;

Compounds 61-62:2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile;

Compound 63:5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)benzonitrile;

Compound 64:1-(5-(4-((2R,7R,8aR)-2-fluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 65:1-(5-(4-((2R,7S,8aR)-2-fluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 66:4-((R/S,S/R)-Octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-cyclopropyl-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 67:1-(5-(4-((2S,7R,8aR)-2-fluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 68:1-(5-(4-((2R,7R,8aR)-2-hydroxy-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compounds 69-70:5-Fluoro-N2-(4-fluoro-3-(5-methyl-1H-tetrazol-1-yl)phenyl)-N4-(octahydro-5,5,8-trimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 69:1-(5-(5-Fluoro-4-(octahydro-5,5,8-trimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 70:5-Fluoro-N2-(4-fluoro-3-(5-methyl-1H-tetrazol-1-yl)phenyl)-N4-(octahydro-5,5,8-trimethylindolizin-7-yl)pyrimidine-2,4-diamine;

Compound 76:4-(R,S)-Octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-cyclopropyl-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 77:1-(2-(2-hydroxyethoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 78:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxyethoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 79:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((S)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 80:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxy-2-methylpropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 81:1-(2-((S)-2-hydroxypropoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 82:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-((R)-2-hydroxypropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 83:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-3-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 84:4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-2-(2-fluoro-4-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-methyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;

Compound 85:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 86:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 87:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(2-hydroxy-2-methylpropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 88:1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;

Compound 89:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one;and

Compound 90:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((R)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimindine-5-carbonitrile;

The present disclosure provides a compound according to the formula:

wherein R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected fromhydrogen, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl,acylamino, acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Z¹ is OR¹⁷, —NR¹⁷R¹⁸ or X;

Z² is H, or Z¹ and Z² together form an oxo, ═NR¹⁹ or ═NNR²⁰R²¹;

R¹⁷ is H, alkyl, substituted alkyl, acyl, acylamino, —SO₂-alkyl or—SO₂-aryl;

R¹⁸ is H, alkyl, substituted alkyl, acyl, acylamino, —SO₂-alkyl,—SO₂-aryl, aryl or heteroaryl;

X is halo or azido;

R¹⁹, R²⁰ and R²¹ each independently are selected from alkyl, substitutedalkyl, aryl substituted aryl, heteroaryl and substituted heteroaryl; and

the compound is optically active.

In certain embodiments, for formula (VI), there is an enantiomericexcess of 90% or more. In certain embodiments, for formula (VI), thereis an enantiomeric excess of 95% or more.

In certain embodiments, in formula (VI), R¹⁸ comprises the formula

wherein R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl; Y¹ and Y² are independently selectedfrom hydrogen and alkyl; and

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl.

In certain embodiments, the compound of formula (VI) comprises theformula

In certain embodiments, the compound of formula (VI) comprises theformula

In certain embodiments, the compound of formula (VI) comprises theformula

In certain embodiments, the compound of formula (VI) comprises theformula

In certain embodiments, the compound of formula (VI) comprises theformula

The compounds described also include isotopically labeled compoundswhere one or more atoms have an atomic mass different from the atomicmass conventionally found in nature. Examples of isotopes that may beincorporated into the compounds disclosed herein include, but are notlimited to, ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, etc. Thus, thedisclosed compounds may be enriched in one or more of these isotopesrelative to the natural abundance of such isotope. By way of example,deuterium (²H) has a natural abundance of about 0.015%. Accordingly, forapproximately every 6,500 hydrogen atoms occurring in nature, there isone deuterium atom. Specifically contemplated herein are compoundsenriched in deuterium at one or more positions. Thus, deuteriumcontaining compounds of the disclosure have deuterium at one or morepositions (as the case may be) in an abundance of greater than 0.015%.

The present disclosure also provides pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound of formula I-V or a pharmaceuticallyacceptable salt or solvate or stereoisomer thereof.

A disclosed compound can be administered alone, as the sole activepharmaceutical agent, or in combination with one or more additionalcompounds of formula I-V or in conjunction with other agents. Whenadministered as a combination, the therapeutic agents can be formulatedas separate compositions that are administered simultaneously or atdifferent times, or the therapeutic agents can be administered togetheras a single composition combining two or more therapeutic agents. Thus,the pharmaceutical compositions disclosed herein containing a compoundof formula I-V optionally contain other therapeutic agents. Accordingly,certain embodiments are directed to such pharmaceutical compositions,wherein the composition further comprises a therapeutically effectiveamount of an agent selected as is known to those of skill in the art.

Since subject compounds possess PKC inhibitory properties, suchcompounds are also useful as research tools. Accordingly, the disclosurealso provides for a method for using a compound of formula I-V or a saltor solvate or stereoisomer thereof as a research tool for studying abiological system or sample, or for discovering new chemical compoundshaving PKC inhibitory properties.

The embodiments are also directed to a compound of formula I-V or a saltor solvate or stereoisomer thereof, for use in therapy or as amedicament.

Additionally, the embodiments are directed to the use of a compound offormula I-V or a salt or solvate or stereoisomer thereof, for themanufacture of a medicament; especially for the manufacture of amedicament for the inhibition of protein kinase C (PKC) activity. Theembodiments are also directed to the use of a compound of formula I-V ora salt or solvate or stereoisomer thereof for the manufacture of amedicament for the treatment of a disease or disorder mediated orsustained through the activity of PKC activity. The embodiments are alsodirected to the use of a compound of formula I-V or a salt or solvate orstereoisomer thereof for the manufacture of a medicament for thetreatment of a disease or disorder associated with the activation ofT-cells, including, inflammatory, autoimmune and allergic disorders.

General Synthetic Procedures

Many general references providing commonly known chemical syntheticschemes and conditions useful for synthesizing the disclosed compoundsare available (see, e.g., Smith and March, March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, Fifth Edition,Wiley-Interscience, 2001; or Vogel, A Textbook of Practical OrganicChemistry, Including Qualitative Organic Analysis, Fourth Edition, NewYork: Longman, 1978).

Compounds as described herein can be purified by any of the means knownin the art, including chromatographic means, such as HPLC, preparativethin layer chromatography, flash column chromatography and ion exchangechromatography. Any suitable stationary phase can be used, includingnormal and reversed phases as well as ionic resins. Most typically thedisclosed compounds are purified via silica gel and/or aluminachromatography. See, e.g., Introduction to Modern Liquid Chromatography,2nd Edition, ed. L. R. Snyder and J. J. Kirkland, John Wiley and Sons,1979; and Thin Layer Chromatography, ed E. Stahl, Springer-Verlag, NewYork, 1969.

During any of the processes for preparation of the subject compounds, itmay be necessary and/or desirable to protect sensitive or reactivegroups on any of the molecules concerned. This may be achieved by meansof conventional protecting groups as described in standard works, suchas J. F. W. McOmie, “Protective Groups in Organic Chemistry”, PlenumPress, London and New York 1973, in T. W. Greene and P. G. M. Wuts,“Protective Groups in Organic Synthesis”, Third edition, Wiley, New York1999, in “The Peptides”; Volume 3 (editors: E. Gross and J. Meienhofer),Academic Press, London and New York 1981, in “Methoden der organischenChemie”, Houben-Weyl, 4^(th) edition, Vol. 15/1, Georg Thieme Verlag,Stuttgart 1974, in H.-D. Jakubke and H. Jescheit, “Aminosauren, Peptide,Proteine”, Verlag Chemie, Weinheim, Deerfield Beach, and Basel 1982,and/or in Jochen Lehmann, “Chemie der Kohlenhydrate: Monosaccharide andDerivate”, Georg Thieme Verlag, Stuttgart 1974. The protecting groupsmay be removed at a convenient subsequent stage using methods known fromthe art.

The subject compounds can be synthesized via a variety of differentsynthetic routes using commercially available starting materials and/orstarting materials prepared by conventional synthetic methods. Suitableexemplary methods that can be routinely adapted to synthesize the2,4-pyrimidinediamine compounds and prodrugs of the invention are foundin U.S. Pat. No. 5,958,935, the disclosure of which is incorporatedherein by reference. Specific examples describing the synthesis ofnumerous 2,4-pyrimidinediamine compounds and prodrugs, as well asintermediates therefore, are described in the U.S. publication No.US2004/0029902A1, the contents of which are incorporated herein byreference. Suitable exemplary methods that can be routinely used and/oradapted to synthesize active 2,4-pyrimidinediamine compounds can also befound in WO 03/063794, U.S. application Ser. No. 10/631,029 filed Jul.29, 2003, WO2004/014382, U.S. publication No. 2005-0234049 A1, andWO005/016893, the disclosures of which are incorporated herein byreference. All of the compounds described herein (including prodrugs)can be prepared by routine adaptation of these methods.

Exemplary synthetic methods for the 2,4-substituted pyrimidinediaminesdescribed herein are described below. Those of skill in the art willalso be able to readily adapt these methods for the synthesis ofspecific 2,4-substituted pyrimidinediamines as described herein.

A variety of exemplary synthetic routes that can be used to synthesizethe 2,4-pyrimidinediamine compounds of the invention are described inscheme below. These methods can be routinely adapted to synthesize the2,4-pyrimidinediamine compounds and prodrugs described herein.

Synthesis of Compounds

In a certain embodiment, the compounds can be synthesized fromsubstituted or unsubstituted uracils as illustrated in Scheme 1, below:

In Scheme 1, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R²⁰, Y¹, Y²,Q, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are as set forth hereinbefore.

According to Scheme 1, uracil A-1 is dihalogenated at the 2- and4-positions using a standard dehydrating-halogenating agent such asPOCl₃ (phosphorus oxychloride) (or other standard halogenating agent)under standard conditions to yield 2,4 dichloropyrimidine A-2. Dependingupon the substituents in pyrimidinediamine A-2, the chloride at the C4position is more reactive towards nucleophiles than the chloride at theC2 position. This differential reactivity can be exploited by firstreacting 2,4 dichloropyrimidine A-2 with one equivalent of amine A-3,yielding 4N-substituted-2-chloro-4-pyrimidineamine A-4, followed byamine A-5 to yield a 2,4-pyrimidinediamine derivative A-6.

Typically, the C4 halide is more reactive towards nucleophiles, asillustrated in the scheme. However, as will be recognized by skilledartisans, the identity of the substituent may alter this reactivity. Forexample, when the substituent is trifluoromethyl, a 50:50 mixture of4N-substituted-4-pyrimidineamine A-4 and the corresponding2N-substituted-2-pyrimidineamine is obtained. The regioselectivity ofthe reaction can also be controlled by adjusting the solvent and othersynthetic conditions (such as temperature), as is well-known in the art.

In a certain embodiment, to couple compounds with an electrophilicleaving group, such as halides or pseudohalides, and compounds with anamino group, nucleophilic aromatic substitution can be used. Forexample, a halogen substituent on Compound A-2 and the amino group onCompound A-3 can react. Also for example, a halogen substituent onCompound A-4 and the amino group on Compound A-5 can react. Conditionsfor nucleophilic aromatic substitution include the compounds reacting ina polar aprotic solvent or polar protic solvent. Suitable solventsinclude alcohols (such as isopropanol, methanol, ethanol), formic acid,dimethylsulfoxide, dimethylformamide, dioxane, and tetrahydrofuran. Thereaction can be run at room temperature or can be heated.

In a certain embodiment, to couple compounds with an electrophilicleaving group, such as halides or pseudohalides, and aryl compounds withan amino group, a coupling reaction, such as a Buchwald couplingreaction, can be used. The Buchwald coupling reaction involvespalladium-catalyzed synthesis of aryl amines. Starting materials arearyl halides or pseudohalides (for example, triflates) and primary orsecondary amines. Such reaction can be performed using a variety ofmethods well known in the art and specific examples can be had byreference to the Examples hereunder described.

The reactions depicted in Scheme 1 may proceed more quickly when thereaction mixtures are heated via microwave. When heating in thisfashion, the following conditions can be used: heat to 175° C. inethanol for 5-20 minutes in a Smith Reactor (Personal Chemistry,Uppsala, Sweden) in a sealed tube (at 20 bar pressure).

A specific embodiment of Scheme 1 utilizing 5-fluorouracil (Aldrich#32,937-1) as a starting material is illustrated in Scheme 2, below.

In Scheme 2, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R²⁰, Y¹, Y²,Q, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are as set forth hereinbefore.

Asymmetric 2N,4N-disubstituted-5-fluoro-2,4-pyrimidinediamine A-10 canbe obtained by reacting 2,4-dichloro-5-fluoropyrimidine A-8 with oneequivalent of amine A-3 (to yield2-chloro-N4-substituted-5-fluoro-4-pyrimidineamine A-9) followed by oneor more equivalents of amine A-5.

A specific embodiment of Scheme 1 to form cyano derivatives isillustrated in Scheme 3, below.

In Scheme 3, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R²⁰, Y¹, Y²,Q, R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ are as set forth hereinbefore.

Asymmetric 2N,4N-disubstituted-5-cyano-2,4-pyrimidinediamine A-15 can beobtained by reacting 2,4-dichloro-5-carbamoylpyrimidine A-12 with oneequivalent of amine A-3 (to yield2-chloro-N4-substituted-5-carbamoyl-4-pyrimidineamine A-13). The amidegroup of Compound A-13 is converted to a cyano group to yield CompoundA-14, followed by reaction with one or more equivalents of amine A-5.Conversion of the amide group to the cyano group can be accomplishedwith dehydration, such as with use of Burgess reagent or trifluoroaceticanhydride. As will be recognized by those of skill in the art andexemplified herein, aniline A-5 may also be reacted with intermediateA-13, and the resultant N2,N4-disubstituteddiaminopyrimidine-5-carbamoylpyrimidine can be dehydrated to yield thecorresponding 5-cyano compound A-15.

Uracil Starting Materials and Intermediates

The uracil A-1, A-7, and A-11 starting materials can be purchased fromcommercial sources or prepared using standard techniques of organicchemistry. Commercially available uracils that can be used as startingmaterials in the schemes disclosed herein include, by way of example andnot limitation, uracil (Aldrich #13,078-8; CAS Registry 66-22-8); 5bromouracil (Aldrich #85,247-3; CAS Registry 51-20-7; 5 fluorouracil(Aldrich #85,847-1; CAS Registry 51-21-8); 5 iodouracil (Aldrich#85,785-8; CAS Registry 696-07-1); 5 nitrouracil (Aldrich #85,276-7; CASRegistry 611-08-5); 5 (trifluoromethyl)-uracil (Aldrich #22,327-1; CASRegistry 54-20-6). Additional 5-substituted uracils are available fromGeneral Intermediates of Canada, Inc., Edmonton, Calif. and/orInterchim, Cedex, France, or can be prepared using standard techniques.Myriad textbook references teaching suitable synthetic methods areprovided infra.

Amino Starting Materials and Intermediates

Amines, such as A-3 and A-5 can be purchased from commercial sources or,alternatively, can be synthesized utilizing standard techniques. Forexample, suitable amines can be synthesized from nitro precursors usingstandard chemistry. See also Vogel, 1989, Practical Organic Chemistry,Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc. By way ofexample amines A-5 can be prepared as described in WO 2010/090875, WO2010-083207, WO 2011/068898 and WO 2012/012619, each of which areincorporated herein by reference.

In a certain embodiment, Compound A-3 can be synthesized as illustratedin Scheme 4, below:

Referring to Scheme 4, amine Compound B-1 reacts with enone Compound B-2to form Compound B-3. First, the condensation reaction between CompoundB-1 and Compound B-2 occurs by mixing the compounds neat or in asolvent. Then, the intermediate from the reaction between Compound B-1and Compound B-2 is formed. The protecting groups are removed fromintermediate. For example and not meant to be limiting, Scheme 4 shows adiethyl acetal as a protecting group. Conditions to remove the diethylacetal protecting group include refluxing the intermediate in aqueousacid. After the protecting group is removed, further reaction affordsCompound B-3.

With continued reference to Scheme 4, the carbonyl group of Compound B-3reacts with hydroxylamine to form Compound B-4. Then, reduction of theoxime group of Compound B-4 affords Compound A-3. Suitable conditionsfor reduction of the oxime group include hydrogenation or reaction withsodium amalgam, borane, or sodium cyanoborohydride. In certainembodiments, the oxime group is hydrogenated with a catalyst. Suitablehydrogenation catalysts include PtO₂ and Raney Ni.

The isomers of Compound A-3 can be isolated by procedures known to thoseskilled in the art. The individual isomers may be obtained, forinstance, by a resolution technique or by chiral chromatographytechniques.

In a certain embodiment, Compound A-3 can be synthesized as illustratedin Scheme 5, below:

Referring to Scheme 5, amine Compound B-1 reacts with enone Compound B-2to form Compound B-3. First, the condensation reaction between CompoundB-1 and Compound B-2 occurs by mixing the compounds neat or in asolvent. Then, the intermediate from the reaction between Compound B-1and Compound B-2 is formed. The protecting groups are removed fromintermediate. For example and not meant to be limiting, Scheme 5 shows adiethyl acetal as a protecting group. Conditions to remove the diethylacetal protecting group include refluxing the intermediate in aqueousacid. After the protecting group is removed, further reaction affordsCompound B-3.

With continued reference to Scheme 5, the carbonyl of Compound B-3 isreduced to afford Compound B-5. Suitable conditions for reduction of thecarbonyl group include hydrogenation or reaction with a metal hydride.In certain embodiments, the carbonyl group is reduced with metalhydride, such as L-selectride ((sec-Bu)BH)₃BHLi), sodium borohydride,lithium aluminum hydride, borane, or aluminum hydride.

Next, the stereoisomers of Compound B-5 are separated. The isomers ofCompound B-5 can be isolated by procedures known to those skilled in theart. The individual isomers may be obtained, for instance, by aresolution technique or by chiral chromatography techniques. In certainembodiments, the stereoisomers of Compound B-5 can be isolated with thehelp of enzyme-catalyzed acylation. Acylation of one of the isomers isperformed to differentiate the isomers. Suitable enzymes include Novozym325 (Aldrich) and subtilisin. For the acylation reagent, an acetatesubstrate, such as vinylacetate or ethyl acetate, can be used.

Next, for example and not meant to be limiting, Compound B-7 is obtainedfor further reaction. Alternatively, Compound B-6 can also be obtainedfor further reaction, depending on which stereoisomer is desired.However, Compound B-7 is shown in the above scheme for further reactionfor the purposes of being an example. With continued reference to Scheme5, the hydroxyl group of Compound B-7 is converted to a leaving group(—O-LG¹) to form Compound B-8. Examples of leaving groups includenonaflates, triflates, fluorosulfonates, tosylates, and mesylates. Incertain embodiments, the hydroxyl group of Compound B-7 is converted toa tosylate or mesylate. Alternatively the transformation of B-7 to B-9can be accomplished as is known to those of skill in the art via in situactivation of the B-7 hydroxyl group, such as via a Mitsonobu reaction.

Then, the leaving group of Compound B-8 is displaced with an azide (—N₃)to form Compound B-9. Reaction of Compound B-8 with sodium azide canform Compound B-9. Then, conversion of the azide of Compound B-9 to forman amine of Compound B-10 can be performed with reduction. Azides may bereduced to amines by hydrogenolysis or with a phosphine, e.g.triphenylphosphine, in the Staudinger reaction. Hydrogenolysisconditions include reaction with hydrogen and a catalyst, such asPd(OH)₂ or Pd/C.

Although many of the synthetic schemes discussed above do not illustratethe use of protecting groups, skilled artisans will recognize that insome instances certain substituents may include functional groupsrequiring protection. The exact identity of the protecting group usedwill depend upon, among other things, the identity of the functionalgroup being protected and the reaction conditions used in the particularsynthetic scheme, and will be apparent to those of skill in the art.Guidance for selecting protecting groups, their attachment and removalsuitable for a particular application can be found, for example, inGreene & Wuts, supra.

Prodrugs as described herein can be prepared by routine modification ofthe above-described methods. Alternatively, such prodrugs can beprepared by reacting a suitably protected 2,4-pyrimidinediamine with asuitable progroup. Conditions for carrying out such reactions and fordeprotecting the product to yield prodrugs as described herein arewell-known.

Myriad references teaching methods useful for synthesizing pyrimidinesgenerally, as well as starting materials described in Schemes (I)-(VII),are known in the art. For specific guidance, the reader is referred toBrown, D. J., “The Pyrimidines”, in The Chemistry of HeterocyclicCompounds, Volume 16 (Weissberger, A., Ed.), 1962, IntersciencePublishers, (A Division of John Wiley & Sons), New York (“Brown I”);Brown, D. J., “The Pyrimidines”, in The Chemistry of HeterocyclicCompounds, Volume 16, Supplement I (Weissberger, A. and Taylor, E. C.,Ed.), 1970, Wiley-Interscience, (A Division of John Wiley & Sons), NewYork (Brown II”); Brown, D. J., “The Pyrimidines”, in The Chemistry ofHeterocyclic Compounds, Volume 16, Supplement II (Weissberger, A. andTaylor, E. C., Ed.), 1985, An Interscience Publication (John Wiley &Sons), New York (“Brown III”); Brown, D. J., “The Pyrimidines” in TheChemistry of Heterocyclic Compounds, Volume 52 (Weissberger, A. andTaylor, E. C., Ed.), 1994, John Wiley & Sons, Inc., New York, pp. 1-1509(Brown IV″); Kenner, G. W. and Todd, A., in Heterocyclic Compounds,Volume 6, (Elderfield, R. C., Ed.), 1957, John Wiley, New York, Chapter7 (pyrimidines); Paquette, L. A., Principles of Modern HeterocyclicChemistry, 1968, W. A. Benjamin, Inc., New York, pp. 1-401 (uracilsynthesis pp. 313, 315; pyrimidinediamine synthesis pp. 313-316; aminopyrimidinediamine synthesis pp. 315); Joule, J. A., Mills, K. and Smith,G. F., Heterocyclic Chemistry, 3rd Edition, 1995, Chapman and Hall,London, UK, pp. 1-516; Vorbrüggen, H. and Ruh-Pohlenz, C., Handbook ofNucleoside Synthesis, John Wiley & Sons, New York, 2001, pp. 1-631(protection of pyrimidines by acylation pp. 90-91; silylation ofpyrimidines pp. 91-93); Joule, J. A., Mills, K. and Smith, G. F.,Heterocyclic Chemistry, 4th Edition, 2000, Blackwell Science, Ltd,Oxford, UK, pp. 1-589; and Comprehensive Organic Synthesis, Volumes 1-9(Trost, B. M. and Fleming, I., Ed.), 1991, Pergamon Press, Oxford, UK.

The embodiments are also directed to processes and novel intermediatesuseful for preparing compounds of formula I-V or a salt or solvate orstereoisomer thereof.

Accordingly, the present disclosure provides a method for making acompound according to the formula

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl, and

Y¹ is selected from hydrogen and alkyl;

the method comprising contacting a compound of the formula

with a compound of the formula:

The present disclosure provides a method for making a compound accordingto the formula

wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;

R¹¹ is selected from alkyl, substituted alkyl, hydroxy, alkoxy,substituted alkoxy, amino, substituted amino, carboxyl, carboxyl ester,cyano, halogen, acyl, aminoacyl, nitro, alkenyl, substituted alkenyl,alkynyl, and substituted alkynyl;

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl;

Y¹ and Y² are independently selected from hydrogen and alkyl; and

Ar¹ is selected from aryl, substituted aryl, heteroaryl, and substitutedheteroaryl;

the method comprising contacting a compound of the formula

wherein X¹ is a halogen;

with a compound of the formula HNY²Ar¹.

In certain embodiments, in the above methods, the method furthercomprises performing separation of isomers with chiral chromatography.In certain embodiments, in the above methods, the method furthercomprises performing separation of isomers with a resolution technique.

The present disclosure provides a method for preparing an opticallyactive compound comprising contacting a racemic mixture of compounds ofthe formula

with a lipase; wherein

R¹, R², R³, R⁴, R⁵, and R⁶ are independently selected from hydrogen,alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino,acyloxy, amino, substituted amino, aminoacyl, aminoacyloxy,oxyaminoacyl, azido, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,thiol, thioalkoxy, substituted thioalkoxy, aryl, aryloxy, hydroxyamino,alkoxyamino, nitro, —SO-alkyl, —SO-substituted alkyl, —SO-aryl,—SO-heteroaryl, —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-aryl and—SO₂-heteroaryl; or R¹ and R² together form an oxo group; or R³ and R⁴together form an oxo group; or R⁵ and R⁶ together form an oxo group;

R⁷, R⁸, R⁹, and R¹⁰ are independently selected from hydrogen, alkyl,substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy,amino, substituted amino, aminoacyl, aminoacyloxy, oxyaminoacyl, azido,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, thiol, thioalkoxy,substituted thioalkoxy, aryl, aryloxy, hydroxyamino, alkoxyamino, nitro,—SO-alkyl, —SO-substituted alkyl, —SO-aryl, —SO-heteroaryl, —SO₂-alkyl,—SO₂-substituted alkyl, —SO₂-aryl and —SO₂-heteroaryl; or R⁷ and R⁸together form an oxo group; or R⁹ and R¹⁰ together form an oxo group;and

R²⁰ is selected from hydrogen, alkyl, and substituted alkyl.

In one embodiment, the above process further comprises the step offorming a salt of a compound of formula I-V. Embodiments are directed tothe other processes described herein; and to the product prepared by anyof the processes described herein.

Pharmaceutical Compositions

The disclosed compounds are useful, at least, for the inhibition of PKCactivity and the treatment of a disease or disorder that is mediatedthrough the activity of a PKC activity. Accordingly, pharmaceuticalcompositions comprising at least one disclosed compound are alsodescribed herein.

A pharmaceutical composition comprising a subject compound may beadministered to a patient alone, or in combination with othersupplementary active agents. The pharmaceutical compositions may bemanufactured using any of a variety of processes, including, withoutlimitation, conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, and lyophilizing.The pharmaceutical composition can take any of a variety of formsincluding, without limitation, a sterile solution, suspension, emulsion,lyophilisate, tablet, pill, pellet, capsule, powder, syrup, elixir orany other dosage form suitable for administration.

A subject compound may be administered to the host using any convenientmeans capable of resulting in the desired reduction in disease conditionor symptom. Thus, a subject compound can be incorporated into a varietyof formulations for therapeutic administration. More particularly, asubject compound can be formulated into pharmaceutical compositions bycombination with appropriate pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants and aerosols.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 19th Edition, 1995, describes exemplaryformulations (and components thereof) suitable for pharmaceuticaldelivery of disclosed compounds. Pharmaceutical compositions comprisingat least one of the subject compounds can be formulated for use in humanor veterinary medicine. Particular formulations of a disclosedpharmaceutical composition may depend, for example, on the mode ofadministration and/or on the location of the infection to be treated. Insome embodiments, formulations include a pharmaceutically acceptablecarrier in addition to at least one active ingredient, such as a subjectcompound. In other embodiments, other medicinal or pharmaceuticalagents, for example, with similar, related or complementary effects onthe affliction being treated can also be included as active ingredientsin a pharmaceutical composition.

Pharmaceutically acceptable carriers useful for the disclosed methodsand compositions are conventional in the art. The nature of apharmaceutical carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions to be administered can optionally contain minor amounts ofnon-toxic auxiliary substances (e.g., excipients), such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like;for example, sodium acetate or sorbitan monolaurate. Other non-limitingexcipients include, nonionic solubilizers, such as cremophor, orproteins, such as human serum albumin or plasma preparations.

Some examples of materials which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

The disclosed pharmaceutical compositions may be formulated as apharmaceutically acceptable salt of a disclosed compound.Pharmaceutically acceptable salts are non-toxic salts of a free baseform of a compound that possesses the desired pharmacological activityof the free base. These salts may be derived from inorganic or organicacids. Non-limiting examples of inorganic acids suitable for formingsalts with the present compounds are hydrochloric acid, nitric acid,hydrobromic acid, sulfuric acid, hydroiodic acid, and phosphoric acid.Non-limiting examples of suitable organic acids for forming salts withthe present compounds are acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, malonic acid, succinic acid, malic acid,maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid,cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid,methyl sulfonic acid, salicylic acid, formic acid, trichloroacetic acid,trifluoroacetic acid, gluconic acid, asparagic acid, aspartic acid,benzenesulfonic acid, para-toluenesulfonic acid, naphthalenesulfonicacid, 1-hydroxy-2-napthoic acid, and the like. Lists of other suitablepharmaceutically acceptable salts are found in Remington'sPharmaceutical Sciences, 19th Edition, Mack Publishing Company, Easton,Pa., 1995. A pharmaceutically acceptable salt may also serve to adjustthe osmotic pressure of the composition.

A subject compound can be used alone or in combination with appropriateadditives to make tablets, powders, granules or capsules, for example,with conventional additives, such as lactose, mannitol, corn starch orpotato starch; with binders, such as crystalline cellulose, cellulosederivatives, acacia, corn starch or gelatins; with disintegrators, suchas corn starch, potato starch or sodium carboxymethylcellulose; withlubricants, such as talc or magnesium stearate; and if desired, withdiluents, buffering agents, moistening agents, preservatives andflavoring agents. Such preparations can be used for oral administration.

A subject compound can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives. The preparation may also be emulsified or the activeingredient encapsulated in liposome vehicles. Formulations suitable forinjection can be administered by an intravitreal, intraocular,intramuscular, subcutaneous, sublingual, or other route ofadministration, e.g., injection into the gum tissue or other oraltissue. Such formulations are also suitable for topical administration.

In some embodiments, a subject compound can be delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

A subject compound can be utilized in aerosol formulation to beadministered via inhalation. A subject compound can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, a subject compound can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. A subject compound can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectcompound calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for a subject compound depend onthe particular compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

The dosage form of a disclosed pharmaceutical composition will bedetermined by the mode of administration chosen. For example, inaddition to injectable fluids, topical or oral dosage forms may beemployed. Topical preparations may include eye drops, ointments, spraysand the like. Oral formulations may be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).Methods of preparing such dosage forms are known, or will be apparent,to those skilled in the art.

Certain embodiments of the pharmaceutical compositions comprising asubject compound may be formulated in unit dosage form suitable forindividual administration of precise dosages. The amount of activeingredient administered will depend on the subject being treated, theseverity of the affliction, and the manner of administration, and isknown to those skilled in the art. Within these bounds, the formulationto be administered will contain a quantity of the extracts or compoundsdisclosed herein in an amount effective to achieve the desired effect inthe subject being treated.

Each therapeutic compound can independently be in any dosage form, suchas those described herein, and can also be administered in various ways,as described herein. For example, the compounds may be formulatedtogether, in a single dosage unit (that is, combined together in oneform such as capsule, tablet, powder, or liquid, etc.) as a combinationproduct. Alternatively, when not formulated together in a single dosageunit, an individual subject compound may be administered at the sametime as another therapeutic compound or sequentially, in any orderthereof.

Methods of Administration

The subject compounds can inhibit a protein kinase C activity.Accordingly, the subject compounds are useful for treating a disease ordisorder that is mediated through the activity of a PKC activity in asubject. Accordingly, the subject compounds are useful for treating adisease or disorder that is associated with the activation of T-cells ina subject.

The route of administration will be selected according to a variety offactors including, but not necessarily limited to, the condition to betreated, the formulation and/or device used, the patient to be treated,and the like. Routes of administration useful in the disclosed methodsinclude but are not limited to oral and parenteral routes, such asintravenous (iv), intraperitoneal (ip), rectal, topical, ophthalmic,nasal, and transdermal. Formulations for these dosage forms aredescribed herein.

An effective amount of a subject compound will depend, at least, on theparticular method of use, the subject being treated, the severity of theaffliction, and the manner of administration of the therapeuticcomposition. A “therapeutically effective amount” of a composition is aquantity of a specified compound sufficient to achieve a desired effectin a subject (host) being treated. For example, this may be the amountof a subject compound necessary to prevent, inhibit, reduce or relieve adisease or disorder that is mediated through the activity of a PKCactivity in a subject. Ideally, a therapeutically effective amount of acompound is an amount sufficient to prevent, inhibit, reduce or relievea disease or disorder that is mediated through the activity of a PKCactivity in a subject without causing a substantial cytotoxic effect onhost cells.

Therapeutically effective doses (or growth inhibitory amounts) of asubject compound or pharmaceutical composition can be determined by oneof skill in the art, with a goal of achieving local (e.g., tissue)concentrations that are at least as high as the IC₅₀ of an applicablecompound disclosed herein.

An example of a dosage range is from about 0.1 to about 200 mg/kg bodyweight orally in single or divided doses. In particular examples, adosage range is from about 1.0 to about 100 mg/kg body weight orally insingle or divided doses, including from about 1.0 to about 50 mg/kg bodyweight, from about 1.0 to about 25 mg/kg body weight, from about 1.0 toabout 10 mg/kg body weight (assuming an average body weight ofapproximately 70 kg; values adjusted accordingly for persons weighingmore or less than average). For oral administration, the compositionsare, for example, provided in the form of a tablet containing from about50 to about 1000 mg of the active ingredient, particularly about 75 mg,about 100 mg, about 200 mg, about 400 mg, about 500 mg, about 600 mg,about 750 mg, or about 1000 mg of the active ingredient for thesymptomatic adjustment of the dosage to the subject being treated. Inone exemplary oral dosage regimen, a tablet containing from about 500 mgto about 1000 mg active ingredient is administered once (e.g., a loadingdose) followed by administration of ½ dosage tablets (e.g., from about250 to about 500 mg) each 6 to 24 hours for at least 3 days.

The specific dose level and frequency of dosage for any particularsubject may be varied and will depend upon a variety of factors,including the activity of the subject compound, the metabolic stabilityand length of action of that compound, the age, body weight, generalhealth, sex and diet of the subject, mode and time of administration,rate of excretion, drug combination, and severity of the condition ofthe host undergoing therapy.

The present disclosure also contemplates combinations of one or moredisclosed compounds with one or more other agents or therapies useful inthe treatment of a disease or disorder. In certain instances, thedisease or disorder is mediated through the activity of a PKC activityin a subject. In certain instances, the disease or disorder is cellproliferative disorder. For example, one or more disclosed compounds maybe administered in combination with effective doses of other medicinaland pharmaceutical agents, or in combination other non-medicinaltherapies, such as hormone or radiation therapy. The term“administration in combination with” refers to both concurrent andsequential administration of the active agents.

Protein Kinase C

Protein Kinase C

PKC is a family of enzymes that function as serine/threonine kinases.The isoenzymes of PKC differ in their tissue distribution, enzymaticselectivity, requirement for Ca²⁺, and regulation. PKCs play animportant role in cell-cell signaling, gene expression and in thecontrol of cell differentiation and growth.

The subject compound can be a selective inhibitor of PKC, e.g. aninhibitor selective for PKC over one or more other protein kinases, e.g.over one or more tyrosine kinases, for instance, over one or morenon-receptor or receptor tyrosine kinases, e.g. over one or more of PKA,PKB, Abl Met, Src, Ins-R, Flt-3, JAK-2, KDR and/or Ret proteins. Theselective PKC inhibitors may optionally be selective over one or moreserine/threonine kinases, e.g. one or more serine/threonine kinaseswhich do not belong to the CDK family. The subject compounds can exhibita selectivity of at least 10 fold, or 20 fold, or 100 fold for the PKCover one or more other protein kinases, e.g. over one or more tyrosinekinases, e.g. over Flt-3, JAK-2, KDR and/or Ret proteins, or over one ormore serine/threonine kinases which do not belong to the CDK family.

The selectivity of a selective inhibitor of PKC over other proteinkinases may be calculated as the ratio of the IC₅₀ measured for PKC inan assay described herein over the IC₅₀ determined for another kinase.In a certain instance, there is provided a PKC inhibitor for which theratio of the IC₅₀ value as determined in an Allogeneic Mixed LymphocyteReaction (MLR) assay to the IC₅₀ value as determined in a BM assay ishigher than 5, 10, 20, or 30. MLR and BM assays can be done according toknown methods, e.g. mouse or human MLR and BM assays, such as disclosedherein.

The disclosure provides an inhibitor of PKC, which can be anisozyme-selective PKC inhibitor, wherein the subject compound possessesselectivity for the isoforms θ and α of PKC over one or more of theother PKC isoforms. In a certain instance, the subject compoundpossesses selectivity for the isoform θ of PKC over one or more of theother PKC isoforms. In a certain instance, the subject compoundpossesses selectivity for the isoform α of PKC over one or more of theother PKC isoforms. In one embodiment, the disclosed compounds exhibitselectivity for PKC θ and PKC α over at least one PKC isoform.

A subject compound can show a selectivity of at least 10 fold, or 20fold, or 100 fold for the isoforms α or α of PKC over one or more of theother PKC isoforms. Selectivity for the isoforms θ or α of PKC over oneor more of the other PKC isoforms can be measured by comparing the IC₅₀of the subject compound for the isoforms θ or α of PKC to the IC₅₀ ofthe subject compound for the other PKC isoforms. In a certain instance,the selectivity can be determined by calculating the ratio of IC₅₀ ofthe subject compound for the other isoforms of PKC to the IC₅₀ of thesubject compound for θ or a isoforms of PKC. In certain examples subjectcompounds exhibit a selectivity for PKC θ, α or both over another PKCisoform of at least about 2-fold, such as from about 3-fold to about300-fold, from about 10-fold to about 100-fold or from about 5-fold to50-fold. IC₅₀ values are obtained, for example, according to PKC assaysdescribed herein. The subject compounds can show an IC₅₀ value for theisoforms θ or α of PKC of 1 μM or less, such as less than about 300 nM,such as from about 1 nM to about 250 nM, less than 100 nM or even lessthan 10 nM in the assays disclosed herein.

The subject compounds can show a selectivity of the isoforms θ or μ ofPKC over other isoforms of PKC, as well as a selectivity over one ormore of the other protein kinases, e.g. over one or more tyrosinekinases, or over one or more serine/threonine kinases which do notbelong to the CDK-family, e.g. over one or more of PKA, PKB, Abl, Met,Src, Ins-it, Flt-3, JAK-2, KDR and Ret proteins, e.g. over one or moreof Flt-3, JAK-2, KDR and Ret proteins.

Certain isozymes of PKC have been implicated in the mechanisms ofvarious disease states, including, but not necessarily limited to, thefollowing: cancer (PKC α, βI, βII, and δ); cardiac hypertrophy and heartfailure (PKC βI and PKC βII) nociception (PKC γ and ε); ischemiaincluding myocardial infarction (PKC ε and δ); immune response,particularly T-cell mediated (PKC θ and α); and fibroblast growth andmemory (PKC δ and ζ). The role of PKC ε is also implicated in painperception. PKC inhibitors can also be used for treating an oculardisease or disorder involving inflammatory and/or neovascular events.

The subject compounds can be used in the treatment of mammalian(especially human) disease states characterized by aberrant, elevatedactivity of a PKC isozyme in a tissue as compared to non-disease tissueof the same origin. PKC isozymes and disease states and/or biologicalfunctions amenable to therapy by inhibition of activity of the PKCisozyme include, but are not necessarily limited to: PKC α(hyperproliferative cellular diseases, such as cancer); PKC βI and PKCβII (cardiac hypertrophy and heart failure); PKC γ (pain management);PKC δ (ischemia, hypoxia (e.g, such as in myocardial infarction and instroke); apoptosis induced by UV irradiation; and aberrant fibroblastgrowth (e.g., as may occur in wound healing)); PKC ε (pain management,myocardial dysfunction); PKC θ (immune system diseases, particularlythose involving T-cell mediated responses); and PKC ζ (memory andfibroblast growth). PKC theta

PKC θ is expressed predominantly in lymphoid tissue and skeletal muscle.PKC θ is selectively expressed in T-cells and plays a role in matureT-cell activation. It has been shown that PKC θ is involved in T-cellreceptor (TCR)-mediated T-cell activation but inessential duringTCR-dependent thymocyte development. PKC θ, but not other PKC isoforms,translocates to the site of cell contact between antigen-specificT-cells and antigen presenting cells (APC), where it localizes with theTCR in the central core of the T-cell activation. PKC θ, but not the α,ε, or ζ isoenzymes, can selectively activate a FasL promoter-reportergene and upregulate the mRNA or cell surface expression of endogenousFasL. On the other hand, PKC θ and ε can promote T-cell survival byprotecting the cells from Fas-induced apoptosis, and this protectiveeffect was mediated by promoting p90Rsk-dependent phosphorylation ofBCL-2 family member BAD. Thus, PKC θ appears to play a dual regulatoryrole in T-cell apoptosis.

PKC θ inhibitors can find use in the treatment or prevention ofdisorders or diseases mediated by T lymphocytes, for example, autoimmunedisease such as rheumatoid arthritis, psoriasis and lupus erythematosus,and inflammatory and/or allergic diseases such as asthma andinflammatory bowel diseases.

PKC θ is a drug target for immunosuppression in transplantation andautoimmune diseases (Isakov et al. (2002) Annual Review of Immunology,20, 761-794). PCT Publication WO2004/043386 identifies PKC θ as a targetfor treatment of transplant rejection and multiple sclerosis. PKC θ alsoplays a role in inflammatory bowel disease (The Journal of Pharmacologyand Experimental Therapeutics (2005), 313 (3), 962-982), asthma (WO2005062918), and lupus (Current Drug Targets: Inflammation & Allergy(2005), 4 (3), 295-298).

In addition, PKC θ is highly expressed in gastrointestinal stromaltumors (Blay, P. et al. (2004) Clinical Cancer Research, 10, 12, Pt. 1),it has been suggested that PKC θ is a molecular target for treatment ofgastrointestinal cancer (Wiedmann, M. et al. (2005) Current Cancer DrugTargets 5(3), 171).

Experiments induced in PKC θ knock-out mice led to the conclusion thatPKC θ inactivation prevented fat-induced defects in insulin signallingand glucose transport in skeletal muscle (Kim J. et al, 2004, The J. ofClinical Investigation 114 (6), 823). This data indicates PKC θ is atherapeutic target for the treatment of type 2 diabetes, and hence PKC θinhibitors can be useful for treating such disease.

Therapeutic Applications

The subject compounds are useful for treating a disease or disorder thatis mediated through, or exacerbated by, the activity of a PKC in asubject in need of treatment. Also, the compounds are useful fortreating a disease or disorder that is associated with aberrant orotherwise undesirable T cell activation in a subject.

Accordingly, the present disclosure provides methods of treating aninflammatory disease in a subject by administering an effective amountof a subject compound, including a salt or solvate or stereoisomerthereof, so as to treat inflammation. Inflammatory diseases contemplatedfor therapy include those characterized by acute inflammation, chronicinflammation or both.

The present disclosure also provides methods of treating an autoimmunedisease in a subject by administering to the subject an effective amountof a subject compound, including a salt or solvate or stereoisomerthereof, so as to treat the autoimmune disease.

The present disclosure also provides methods of treating an oculardisease or disorder involving inflammatory and/or neovascular events byadministration of a subject compound, including a salt or solvate orstereoisomer thereof, in an effective amount.

Diseases or conditions of interest for treatment according to thepresent disclosure include, but are not limited to, atherosclerosis,cardiac arrhythmia, vascular occlusion due to vascular injury such asangioplasty, restenosis, obesity, syndrome X, impaired glucosetolerance, polycystic ovary syndrome, hypertension, heart failure,chronic obstructive pulmonary disease, CNS diseases such as Alzheimerdisease or amyotrophic lateral sclerosis, Parkinson's disease, bipolardisorder, and can be used to induce axon regeneration. Further thecompounds can be used to treat cancer, infectious diseases such as:AIDS, malaria, such as by blocking the development of cerebral malaria,septic shock or adult respiratory distress syndrome,ischemia/reperfusion injury, myocardial infarction, stroke, gutischemia, renal failure, hemorrhagic shock, and traumatic shock, forexample traumatic brain injury. In one aspect, the present compounds areuseful in treating muscle diseases or disorders, including inflammatorymuscle disease and dystrophic disorders, such as Duchenne musculardystrophy and myotonic muscular dystrophy.

Further diseases or conditions that can be treated with the disclosedcompounds according to the present disclosure include, but are notlimited to, T-cell mediated acute or chronic inflammatory, allergic,autoimmune diseases or disorders, including, without limitationrheumatoid arthritis, osteoarthritis, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, diabetestype I or II and the disorders associated therewith, transplantrejection, graft versus host disease, respiratory diseases, asthma,allergic rhinitis, inflammatory lung injury, inflammatory liver injury,inflammatory glomerular injury, cutaneous manifestations ofimmunologically-mediated disorders or illnesses, such as cutaneouslupus, including discoid lupus, inflammatory and hyperproliferative skindiseases (such as psoriasis, atopic dermatitis, allergic contactdermatitis, irritant contact dermatitis and further eczematousdermatitises, seborrhoeic dermatitis), inflammatory eye diseases (suchas Sjoegren's syndrome, keratoconjunctivitis, uveitis) inflammatorybowel disease, Crohn's disease or ulcerative colitis, Guillain-Barresyndrome, and allergies. The disclosed compounds can be used to treatsymptoms associated with the disorders described above. In particularthe presently disclosed compounds can be used to treat complications ofdiabetes or insulin resistance and conditions associated therewith,including diabetic cardiomyopathy, glucose intolerance, fatty liverdisease, hepatic steatosis, particularly non-alcoholic hepaticsteatosis, and can be used to improve insulin sensitivity. Moreover, thepresent compounds also may be used to improve metabolic efficiency, forexample, they can be used to treat exercise intolerance and intermittentclaudication caused by, for example, peripheral artery disease.

The subject compounds can also be used for preventing or treating ordelaying ocular diseases and disorders involving autoimmune or allergicinflammation and/or neovascularization. Ocular diseases or disordersinvolving inflammatory and/or neovascular events include, but are notlimited to, macular degeneration (AMD), allergic conjunctivitis,diabetic ocular diseases or disorders, including diabetic retinopathy,uveitis, optic neuritis, ocular edema, ocular angiogenesis, ischemicretinopathy, anterior ischemic optic neuropathy, optic neuropathy andneuritis, macular edema, cystoid macular edema (CME), retinal disease ordisorder, such as retinal detachment, retinitis pigmentosa (RP),Stargart's disease, Best's vitelliform retinal degeneration, Leber'scongenital amaurosis and other hereditary retinal degenerations,Sorsby's fundus dystrophy, pathologic myopia, retinopathy of prematurity(ROP), Leber's hereditary optic neuropathy, corneal transplantation orrefractive corneal surgery, keratoconjunctivitis, or dry eye.

Generally, cell proliferative disorders treatable with the subjectcompound disclosed herein relate to any disorder characterized byaberrant cell proliferation. These include various tumors and cancers,benign or malignant, metastatic or non-metastatic. Specific propertiesof cancers, such as tissue invasiveness or metastasis, can be targetedusing the methods described herein. Cell proliferative disorders thatcan be treated using the present compounds include a variety of cancers,including, among others, breast cancer, ovarian cancer, renal cancer,gastrointestinal cancer, including gastrointestinal stromal tumors,Ewing's sarcoma, kidney cancer, bladder cancer, pancreatic cancer, lungcancer, including non-small cell lung cancer, including lung squamouscarcinoma, adenocarcinoma and large cell carcinomas.

In some embodiments, the cell proliferative disorder treated is ahematopoietic neoplasm, which is aberrant growth of cells of thehematopoietic system. Hematopoietic malignancies can have its origins inpluripotent stem cells, multipotent progenitor cells, oligopotentcommitted progenitor cells, precursor cells, and terminallydifferentiated cells involved in hematopoiesis. Some hematologicalmalignancies are believed to arise from hematopoietic stem cells, whichhave the ability for self renewal. For instance, cells capable ofdeveloping specific subtypes of acute myeloid leukemia (AML) upontransplantation display the cell surface markers of hematopoietic stemcells, implicating hematopoietic stem cells as the source of leukemiccells. Blast cells that do not have a cell marker characteristic ofhematopoietic stem cells appear to be incapable of establishing tumorsupon transplantation (Blaire et al., 1997, Blood 89:3104-3112). The stemcell origin of certain hematological malignancies also finds support inthe observation that specific chromosomal abnormalities associated withparticular types of leukemia can be found in normal cells ofhematopoietic lineage as well as leukemic blast cells. For instance, thereciprocal translocation t(9q34; 22q11) associated with approximately95% of chronic myelogenous leukemia appears to be present in cells ofthe myeloid, erythroid, and lymphoid lineage, suggesting that thechromosomal aberration originates in hematopoietic stem cells. Asubgroup of cells in certain types of CML displays the cell markerphenotype of hematopoietic stem cells.

Although hematopoietic neoplasms often originate from stem cells,committed progenitor cells or more terminally differentiated cells of adevelopmental lineage can also be the source of some leukemias. Forexample, forced expression of the fusion protein Bcr/Abl (associatedwith chronic myelogenous leukemia) in common myeloid progenitor orgranulocyte/macrophage progenitor cells produces a leukemic-likecondition. Moreover, some chromosomal aberrations associated withsubtypes of leukemia are not found in the cell population with a markerphenotype of hematopoietic stem cells, but are found in a cellpopulation displaying markers of a more differentiated state of thehematopoietic pathway (Turhan et al., 1995, Blood 85:2154-2161). Thus,while committed progenitor cells and other differentiated cells may haveonly a limited potential for cell division, leukemic cells may haveacquired the ability to grow unregulated, in some instances mimickingthe self-renewal characteristics of hematopoietic stem cells (Passegueet al., Proc. Natl. Acad. Sci. USA, 2003, 100:11842-9).

In some embodiments, the hematopoietic neoplasm treated is a lymphoidneoplasm, where the abnormal cells are derived from and/or display thecharacteristic phenotype of cells of the lymphoid lineage. Lymphoidneoplasms can be subdivided into B-cell neoplasms, T and NK-cellneoplasms, and Hodgkin's lymphoma. B-cell neoplasms can be furthersubdivided into precursor B-cell neoplasm and mature/peripheral B-cellneoplasm. Exemplary B-cell neoplasms are precursor B-lymphoblasticleukemia/lymphoma (precursor B-cell acute lymphoblastic leukemia) whileexemplary mature/peripheral B-cell neoplasms are B-cell chroniclymphocytic leukemia/small lymphocytic lymphoma, B-cell prolymphocyticleukemia, lymphoplasmacytic lymphoma, splenic marginal zone B-celllymphoma, hairy cell leukemia, plasma cell myeloma/plasmacytoma,extranodal marginal zone B-cell lymphoma of MALT type, nodal marginalzone B-cell lymphoma, follicular lymphoma, mantle-cell lymphoma, diffuselarge B-cell lymphoma, mediastinal large B-cell lymphoma, primaryeffusion lymphoma, and Burkitt's lymphoma/Burkitt cell leukemia. T-celland Nk-cell neoplasms are further subdivided into precursor T-cellneoplasm and mature (peripheral) T-cell neoplasms. Exemplary precursorT-cell neoplasm is precursor T-lymphoblastic lymphoma/leukemia(precursor T-cell acute lymphoblastic leukemia) while exemplary mature(peripheral) T-cell neoplasms are T-cell prolymphocytic leukemia T-cellgranular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-celllymphoma/leukemia (HTLV-1), extranodal NK/T-cell lymphoma, nasal type,enteropathy-type T-cell lymphoma, hepatosplenic gamma-delta T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, Mycosisfungoides/Sezary syndrome, Anaplastic large-cell lymphoma, T/null cell,primary cutaneous type, Peripheral T-cell lymphoma, not otherwisecharacterized, Angioimmunoblastic T-cell lymphoma, Anaplastic large-celllymphoma, T/null cell, primary systemic type. The third member oflymphoid neoplasms is Hodgkin's lymphoma, also referred to as Hodgkin'sdisease. Exemplary diagnosis of this class that can be treated with thecompounds include, among others, nodular lymphocyte-predominantHodgkin's lymphoma, and various classical forms of Hodgkin's disease,exemplary members of which are Nodular sclerosis Hodgkin's lymphoma(grades 1 and 2), Lymphocyte-rich classical Hodgkin's lymphoma, Mixedcellularity Hodgkin's lymphoma, and Lymphocyte depletion Hodgkin'slymphoma.

In some embodiments, the hematopoietic neoplasm treated is a myeloidneoplasm. This group comprises a large class of cell proliferativedisorders involving or displaying the characteristic phenotype of thecells of the myeloid lineage. Myeloid neoplasms can be subdivided intomyeloproliferative diseases, myelodysplastic/myeloproliferativediseases, myelodysplastic syndromes, and acute myeloid leukemias.Exemplary myeloproliferative diseases are chronic myelogenous leukemia(e.g., Philadelphia chromosome positive (t(9; 22)(qq34; q11)), chronicneutrophilic leukemia, chronic eosinophilic leukemialhypereosinophilicsyndrome, chronic idiopathic myelofibrosis, polycythemia vera, andessential thrombocythemia. Exemplary myelodysplastic/myeloproliferativediseases are chronic myelomonocytic leukemia, atypical chronicmyelogenous leukemia, and juvenile myelomonocytic leukemia. Exemplarymyelodysplastic syndromes are refractory anemia, with ringedsideroblasts and without ringed sideroblasts, refractory cytopenia(myelodysplastic syndrome) with multilineage dysplasia, refractoryanemia (myelodysplastic syndrome) with excess blasts, 5q-syndrome, andmyelodysplastic syndrome with t(9; 12)(q22; p12) (TEL-Syk fusion; see,e.g., Kuno et al., 2001, Blood 97:1050).

In some embodiments, the composition can be used to treat acute myeloidleukemias (AML), which represent a large class of myeloid neoplasmshaving its own subdivision of disorders. These subdivisions include,among others, AMLs with recurrent cytogenetic translocations, AML withmultilineage dysplasia, and other AML not otherwise categorized.Exemplary AMLs with recurrent cytogenetic translocations include, amongothers, AML with t(8; 21)(q22; q22), AML1(CBF-alpha)/ETO, Acutepromyelocytic leukemia (AML with t(15; 17)(q22; q11-12) and variants,PML/RAR-alpha), AML with abnormal bone marrow eosinophils(inv(16)(p13q22) or t(16; 16)(p13; q11), CBFb/MYH11X), and AML with11q23 (MLL) abnormalities. Exemplary AML with multilineage dysplasia arethose that are associated with or without prior myelodysplasticsyndrome. Other acute myeloid leukemias not classified within anydefinable group include, AML minimally differentiated, AML withoutmaturation, AML with maturation, Acute myelomonocytic leukemia, Acutemonocytic leukemia, Acute erythroid leukemia, Acute megakaryocyticleukemia, Acute basophilic leukemia, and Acute panmyelosis withmyelofibrosis.

In other aspects, cell proliferative disorders comprise virally mediatedtumors. These can arise from infection of cells by an oncogenic virusthat has the capability of transforming a normal cell into a tumor cell.Because rates of viral infection far exceed the number of actualincidence of cell transformation, viral mediated transformationgenerally act together with other cellular factors to generate atransformed tumor cell. Thus, a virally mediated tumor does not requirethe virus to be the sole causative agent of the cell proliferativedisorder, but rather that the viral infection or persistent presence ofvirus is associated with the generation of the tumor. Generally, tumorswhere the causative agent is a virus typically has continual expressionof a limited number of viral genes and that viral these oncogenes,expressed as part of the viral infection or through persistence of thevirus, disrupts the normal cellular gene expression and signaltransduction pathways. Without being bound by theory, viral oncogenesinvolved in cell transformation appear to disrupt four main cellularprocesses: cell surface receptors that interact with growth factors andextracellular matrix, transmembrane signaling networks, cytosolicelements such as soluble proteins and second messengers, and nuclearproteins including DNA binding proteins and factors which functiondirectly and indirectly in gene regulation and replication.

Characterization of Functional Properties

The following are exemplary assays useful in characterizing activitiesof a compound of interest.

A. In Vitro

1. Protein Kinase C assay

The inhibition of PKC activity is measured by monitoring the productionof phosphorylated peptide by fluorescence polarization at differentconcentrations of the inhibitor. Reactions are carried out in 96-wellplate format with a total volume of 20 μL, containing 20 mM HEPES, pH7.4, 5 mM MgCl₂, 0.2 mM CaCl₂, 1 mM DTT, 0.02% Brij-35, 0.1 mg/mlphosphatidylserine, 0.02 mg/ml dioleoyl-sn-glycerol and 5 μM each of ATPand the peptide substrate. Compounds are first diluted serially in DMSOand then transferred to a solution containing the above concentrationsof HEPES, MgCl₂, CaCl₂, DTT, and Brij-35 to yield 5× compound solutionsin 2% DMSO, which is then added to the reaction solution. Reactions areinitiated by the addition of PKC at a typical concentration as describedin the table below, and then allowed to incubate at room temperature for20 minutes. At the end of this time, a combination of quench (EDTA) anddetection (peptide tracer and antibody) reagents is added using theprotocol of Invitrogen P2748 (Carlsbad, Calif.), a Protein Kinase CFluorescence polarization Assay Kit. After a 30 minute period ofincubation, the amount of phosphorylated peptide generated is measuredby fluorescence polarization (Ex=485 nm, Em=535 nm) using a TecanPolarian instrument (Switzerland).

TABLE 2 enzyme Peptide substrate SEQ ID Enzyme source concentration PKCRFARKGSLRQKNV Seq ID No. 1 Upstate 40 ng/ml theta Biotechnologies,Temecula, CA, cat. #14-444 PKC RFARKGSLRQKNV Seq ID No. 1 Upstate 50ng/ml epsilon Biotechnologies, Temecula, CA, cat. #14-5182. IL-2 ELISA, Human Primary T Cell, Anti-CD3+CD28+ Assays

Human Primary T Cell Isolation and Culture:

Human primary T cells were prepared as follows. Fresh PBMC's from AllCells (Cat # PB002) were re-suspended in RPMI (RPMI-1640 withL-Glutamine; Mediatech, Inc., Herndon Va., cat. #10-040-CM) with 10% FBSand seeded into flasks and incubated at 37° C. for 2 hours to allow themonocytes to adhere. The non-adherent cells were then centrifuged andre-suspended in RPMI medium containing 40 U/ml IL2 and seeded into aflask pre-coated with 1 μg/ml aCD3 and 5 ug/ml aCD28 (Anti-Human CD3, BDPharmingen Catalog #555336, Anti-Human CD28, Beckman Coulter Catalog#IM1376). The cells were stimulated for 3-4 days, then transferred to afresh flask and maintained in RPMI (RPMI-1640 with L-Glutamine;Mediatech, Inc., Herndon Va., cat. #10-040-CM) with 10% FBS and 40 U/mlIL-2.

Primary T Cell Stimulation and IL2 ELISA:

Human primary T cells (100,000 cells per well) were pre-incubated withor without test compound in RPMI-1640 with L-Glutamine and 10% FBS for 1hour at 37° C. Cells were then stimulated by transferring them toround-bottom 96-well plates pre-coated with 1 μg/ml aCD3 and 5 μg/mlaCD28. For counter assay, cells were instead stimulated by adding 8×stock solutions of PMA and ionomycin in RPMI-1640 with L-Glutamine and10% FBS (for final concentrations of 0.5 ng/ml PMA and 0.1 μM ionomycin,both from Calbiochem). Cells were incubated at 37° C. for 24 hoursbefore 100 μL supernatants were harvested for quantification of IL-2 byELISA using Human IL-2 Duoset ELISA Kit from R and D Systems, Cat. #DY202E.

3. Protein Kinase C Assay

The subject compounds can be tested for activity on different PKCisoforms according to the following method. Assay is performed in awhite with clear bottom 384-well microtiterplate with non-bindingsurface. The reaction mixture (25 μl) contains 1.5 μM of atridecapeptide acceptor substrate that mimics the pseudo substratesequence of PKC α with the Ala→Ser replacement, 10 μM ³³P-ATP, 10 mMMg(NO₃)₂, 0.2 mM CaCl₂, PKG at a protein concentration varying from 25to 400 ng/ml (depending on the isotype used), lipid vesicles (containing30 mol % phosphatidylserine, 5 mol % DAG and 65 mol %phosphatidylcholine) at a final lipid concentration of 0.5 mM, in 20 mMTris-HCl buffer pH 7.4+0.1% BSA. Incubation is performed for 60 minutesat room temperature. Reaction is stopped by adding 50 μl of stop mix(100 mM EDTA, 200 μM ATP, 0.1% Triton X-100, 0.375 mg/wellstreptavidin-coated SPA beads in phosphate buffered saline w/o Ca, Mg.After 10 minutes incubation at room temperature, the suspension is spundown for 10 minutes at 300 g. Incorporated radioactivity is measured ina Trilux counter for 1 minute. IC₅₀ measurement is performed on aroutine basis by incubating a serial dilution of inhibitor atconcentrations ranging between 1-1000 μM. IC₅₀ values are calculatedfrom the graph by curve fitting with XL Fit® software.

4. Protein Kinase C α Assay

Human recombinant PKC α is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

5. Protein Kinase C β1 Assay

Human recombinant PKC β1 is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

6. Protein Kinase C δ Assay

Human recombinant PKC δ is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

7. Protein Kinase C & Assay

Human recombinant PKC ε is obtained from Oxford Biomedical Research andis used under the assay conditions as described under Section A.1 above.

8. Protein Kinase C η Assay

Human recombinant PKC η is obtained from PanVera and is used under theassay conditions as described under Section A.1 above.

9. Protein Kinase C θ Assay

Human recombinant PKC θ is used under the assay conditions as describedabove.

10. CD28 Costimulation Assay

The assay is performed with Jurkat cells transfected with a humaninterleukin-2 promoter/reporter gene construct as described by Baumann Get al. in Transplant. Proc. 1992; 24:43-8, the β-galactosidase reportergene being replaced by the luciferase gene (de Wet J., et al., Mol.Cell. Biol. 1987, 7(2), 725-737). Cells are stimulated by solidphase-coupled antibodies or phorbol myristate acetate (PMA) and theCa⁺⁺ionophore ionomycin as follows. For antibody-mediated stimulationMicrolite TM1 microtiter plates (Dynatech) are coated with 3 μg/ml goatanti-mouse IgG Fc antibodies (Jackson) in 55 μl phosphate-bufferedsaline (PBS) per well for three hours at room temperature. Plates areblocked after removing the antibodies by incubation with 2% bovine serumalbumin (BSA) in PBS (300 μl per well) for 2 hours at room temperature.After washing three times with 300 μl PBS per well, 10 ng/ml anti-T cellreceptor antibodies (WT31, Becton & Dickinson) and 300 ng/ml anti-CD28antibodies (15E8) in 50 μl 2% BSA/PBS are added as stimulatingantibodies and incubated overnight at 4° C. Finally the plates arewashed three times with 300 μl PBS per well. Seven three-fold serialdilutions of test compounds in duplicates in assay medium (RPMI 1640/10%fetal calf serum (FCS) containing 50 μM 2-mercaptoethanol, 100 units/mlpenicillin and 100 μg/ml streptomycin) are prepared in separate plates,mixed with transfected Jurkat cells (clone K22 290_H23) and incubatedfor 30 minutes at 37° C. in 5% CO₂ 100 μl of this mixture containing1×10⁵ cells are then transferred to the antibody-coated assay plates. Inparallel 100 μl are incubated with 40 ng/ml PMA and 2 μM ionomycin.After incubation for 5.5 hours at 37° C. in 5% CO₂, the level ofluciferase is determined by bioluminescence measurement. The plates arecentrifuged for 10 minutes at 500 g and the supernatant is removed byflicking. Lysis buffer containing 25 mM Tris-phosphate, pH 7.8, 2 mMDTT, 2 mM 1,2-diaminocyclohexane-N,N,N′,N-tetraacetic acid, 10% (v/v)glycerol and 1% (v/v) Triton X-100 is added (20 μl per well). The platesare incubated at room temperature for 10 minutes under constant shaking.Luciferase activity is assessed with a bioluminescence reader(Labsystem, Helsinki, Finland) after automatic addition of 50 μl perwell luciferase reaction buffer containing 20 mM Tricine, 1.07 mM(MgCO₃)₄Mg(OH)₂×5H₂O, 2.67 mM MgSO₄, 0.1 mM EDTA, 33.3 mM DTT, 270 μMcoenzyme A, 470 μM luciferin (Chemie Brunschwig AG), 530 μM ATP, pH 7.8.Lag time is 0.5 seconds, total measuring time is 1 or 2 seconds. Lowcontrol values are light units from anti-T cell receptor- orPMA-stimulated cells, high controls are from anti-T cellreceptor/anti-CD28- or PMA/ionomycin-stimulated cells without any testsample. Low controls are subtracted from all values. The inhibitionobtained in the presence of a test compound is calculated as percentinhibition of the high control. The concentration of test compoundsresulting in 50% inhibition (IC₅₀) is determined from the dose-responsecurves.

11. Bone Marrow Proliferation (BM) Assay

Bone marrow cells from CBA mice (2.5×104 cells per well in flat bottomtissue culture microtiter plates) are incubated in 100 μl RPMI mediumcontaining 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin (GibcoBRL, Basel, Switzerland), 50 UM 2-mercaptoethanol (Fluke, Buchs,Switzerland), WEHI-3 conditioned medium (7.5% v/v) and L929 conditionedmedium (3% v/v) as a source of growth factors and serially dilutedcompounds. Seven three-fold dilution steps in duplicates per testcompound are performed. After four days of incubation 1 μCi³H-thymidineis added. Cells are harvested after an additional five-hour incubationperiod, and incorporated ³H-thymidine is determined according tostandard procedures. Conditioned media are prepared as follows. WEHI-3cells 1 (ATCC TIB68) and L929 cells (ATCC CCL 1) are grown in RPMImedium until confluence for 4 days and one week, respectively. Cells areharvested, resuspended in the same culture flasks in medium C containing1% FCS (Schreier and Tees 1981) for WEHI-3 cells and RPMI medium forL929 cells and incubated for 2 days (WEHI-3) or one week (L929). Thesupernatant is collected, filtered through 0.2 μm and stored in aliquotsat −80° C. Cultures without test compounds and without WEHI-3 and L929supernatants are used as low control values. Low control values aresubtracted from all values. High controls without any sample are takenas 100% proliferation. Percent inhibition by the samples is calculatedand the concentrations required for 50% inhibition (IC₅₀ values) aredetermined.

12. Allogeneic Mixed Lymphocyte Reaction (MLR)

The two-way MLR is performed according to standard procedures (J.Immunol. Methods, 1973, 2, 279 and Meo T. et al., Immunological Methods,New York, Academic Press, 1979, 227-39). Briefly, spleen cells from CBAand BALB/c mice (1.6×10⁵ cells from each strain per well in flat bottomtissue culture microtiter plates, 3.2×10⁵ in total) are incubated inRPMI medium containing 10% FCS, 100 U/ml penicillin, 100 μg/mlstreptomycin (Gibco BRL, Basel, Switzerland), 50 μM 2-mercaptoethanol(Fluka, Buchs, Switzerland) and serially diluted compounds. Seventhree-fold dilution steps in duplicates per test compound are performed.After four days of incubation 1 μCi³H-thymidine is added. Cells areharvested after an additional five-hour incubation period, andincorporated ³H-thymidine is determined according to standardprocedures. Background values (low control) of the MLR are theproliferation of BALB/c cells alone. Low controls are subtracted fromall values. High controls without any sample are taken as 100%proliferation. Percent inhibition by the samples is calculated, and theconcentrations required for 50% inhibition (IC₅₀ values) are determined.

B. In Vivo

Heart Transplantation Model

The strain combination used: Male Lewis (RT¹haplotype) and BN(RT¹haplotype). The animals are anaesthetised using inhalationalisofluorane. Following heparinisation of the donor rat through theabdominal inferior vena cava with simultaneous exsanguination via theaorta, the chest is opened and the heart rapidly cooled. The aorta isligated and divided distal to the first branch and the brachiocephalictrunk is divided at the first bifurcation. The left pulmonary artery isligated and divided and the right side divided but left open. All othervessels are dissected free, ligated and divided and the donor heart isremoved into iced saline.

The recipient is prepared by dissection and cross-clamping of theinfra-renal abdominal aorta and vena cava. The graft is implanted withend-to-side anastomoses, using 1010 monofilament suture, between thedonor brachiocephalic trunk and the recipient aorta and the donor rightpulmonary artery to the recipient vena cava. The clamps are removed, thegraft tethered retroabdominally, the abdominal contents washed with warmsaline and the animal is closed and allowed to recover under a heatinglamp. Graft survival is monitored by daily palpation of the beatingdonor heart through the abdominal wall. Rejection is considered to becomplete when-heart beat stops. Graft survival is monitored in animalstreated with compounds.

Graft v. Host Model

Spleen cells (2×10⁷) from Wistar/F rats are injected subcutaneously intothe right hind footpad of (Wistar/F×Fischer 344)F₁ hybrid rats. The leftfootpad is left untreated. The animals are treated with the testcompounds on 4 consecutive days (0-3). The popliteal lymph nodes areremoved on day 7, and the weight differences between two correspondinglymph nodes are determined. The results are expressed as the inhibitionof lymph node enlargement (given in percent) comparing the lymph nodeweight differences in the experimental groups to the weight differencebetween the corresponding lymph nodes from a group of animals leftuntreated with a test compound. In certain instances the test compoundis a selective PKC inhibitor. For example, disclosed compounds that areparticularly useful for treating graft versus host disease and relateddisorders are selective PKC α and θ inhibitors.

Rat Collagen-Induced Arthritis Model

Rheumatoid arthritis (RA) is characterized by chronic joint inflammationeventually leading to irreversible cartilage destruction. IgG-containingIC are abundant in the synovial tissue of patients with RA. While it isstill debated what role these complexes play in the etiology andpathology of the disease, IC communicate with the hematopoetic cells viathe FcγR.

CIA is a widely accepted animal model of RA that results in chronicinflammatory synovitis characterized by pannus formation and jointdegradation. In this model, intradermal immunization with native type IIcollagen, emulsified with incomplete Freund's adjuvant, results in aninflammatory polyarthritis within 10 or 11 days and subsequent jointdestruction in 3 to 4 weeks.

Study Protocol

Syngeneic LOU rats are immunized with native type II collagen on Day 0,and efficacy of a test compound is evaluated in a prevention regimen anda treatment regimen. In the prevention protocol, either vehicle orvarious doses of a test compound are administered via oral gavagestarting on day of immunization (Day 0). In the treatment protocol,after clinical signs of arthritis develop on Day 10, treatment with atest compound is initiated (e.g., 300 mg/kg by oral gavage, qd) andcontinued until sacrifice on Day 28. In both protocols, clinical scoresare obtained daily, and body weights are measured twice weekly. At Day28, radiographic scores are obtained, and serum levels of collagen IIantibody are measured by ELISA.

Determination of Results

By 10 days after immunization, rats can develop clinical CIA, asdetermined by an increase in their arthritis scores. The mean arthriticscore gradually increases in the rats treated with vehicle alone afterDay 10, and by Day 28 the mean clinical score can reach about 6.75. Meanclinical scores in animals treated from the day of immunization (Day 0)with a test compound can be significantly reduced on Days 10-28 comparedwith vehicle controls. In the rats treated with a test compound atdisease onset, there can be a significantly lower arthritis scorebeginning around Day 16, and this difference can be observed until theend of the study on Day 28.

Blinded radiographic scores (scale 0-6) can be obtained on Day 28 of CIAand compared between the animals in the vehicle group, animals in theprevention group, and animals in the treatment group.

The groups administered with a test compound, either prophylactically(at immunization) or after disease onset can preclude the development oferosions and reduced soft tissue swelling. Similarly, the groupsadministered with a test compound can result in reduction of serumanti-collagen II antibody.

Mouse Experimental Autoimmune Encephalomyelitis

The in vivo efficacy of a test compound towards autoimmune diseases canbe demonstrated in a mouse model of experimental autoimmuneencephalomyelitis (EAE).

Model Description

EAE is a useful model for multiple sclerosis (MS), an autoimmune diseaseof the CNS that is caused by immune-cell infiltration of the CNS whitematter. Inflammation and subsequent destruction of myelin causeprogressive paralysis. Like the human disease, EAE is associated withperipheral activation of T cells autoreactive with myelin proteins, suchas myelin basic protein (MBP), proteolipid protein (PLP), or myelinoligodendrocyte protein (MOG). Activated neuroantigen-specific T cellspass the blood-brain barrier, leading to focal mononuclear cellinfiltration and demyelination. EAE can be induced in susceptible mousestrains by immunization with myelin-specific proteins in combinationwith adjuvant. In the SJL mouse model used in these studies, hind limband tail paralysis is apparent by Day 10 after immunization, the peak ofdisease severity can be observed between Days 10 and 14, and a cycle ofpartial spontaneous remission followed by relapse can be observed up toDay 35. The results can demonstrate the potential of the test compoundto suppress disease severity and prevent relapse of disease symptomsthat may be the result of FcγR-mediated cytokine release from immunecells.

Study Protocol

In the SJL murine model of EAE, each mouse is sensitized with PLP/CFA.(150 μg PLP139-151 with 200 μg CFA in 0.05 ml of homogenate on foursites of hind flank for a total of 0.2 ml emulsion is used to induceEAE). In a suppression protocol, either vehicle or various doses of atest compound are administered via oral gavage starting on the day ofimmunization (Day 0). In a treatment protocol, at onset of disease,animals are separated to achieve groups with a similar mean clinicalscore at onset and administered vehicle or various dose frequencies oftest compounds via oral gavage. In both protocols, clinical scores aremonitored daily, and body weights are measured twice weekly.

Determination of Results

By 10 days after PLP immunization, SJL mice can develop clinical EAE, asevidenced by an increase in their mean clinical scores. The paralyticscore can gradually increase in the animals treated with vehicle onlyfrom the day of immunization (Day 0), and by Day 14 the mean score canreach a peak of about 5.1. At disease peak (e.g., Day 14), the meanclinical score in animals treated with either daily or twice daily canbe significantly reduced. By Day 16, animals can exhibit a partialremission of mean clinical severity, which is a characteristic of theSJL model. The lower clinical scores in animals treated twice daily witha test compound can remain significant throughout the experiment untilthe animals are sacrificed on Day 30. These lower scores throughout thetreatment period are reflected in the significantly lower cumulativedisease index (CDI) and increase in cumulative weight index (CWI).

SJL mice treated with a test compound at disease onset (e.g., Day 11)can show a significant decrease in CDI. Further, there can be a decreasein the number of relapses in animals treated with a test compoundcompared with the number of relapses in animals treated with vehicle.

Research Applications

Since subject compounds can inhibit a PKC activity, such compounds arealso useful as research tools. The present disclosure also provides amethod for using subject compounds as a research tool for studying abiological system or sample, or for discovering new chemical compoundsthat can inhibit a PKC activity.

The disclosure provides for a method of studying a biological system orsample known to comprise PKC, the method comprising: (a) contacting thebiological sample with a compound of formula I-V or a salt or solvate orstereoisomer thereof; and (b) determining the inhibiting effects causedby the compound on the biological sample.

Any suitable biological sample having PKC can be employed in suchstudies which can be conducted either in vitro or in vivo.Representative biological samples suitable for such studies include, butare not limited to, cells, cellular extracts, plasma membranes, tissuesamples, isolated organs, mammals (such as mice, rats, guinea pigs,rabbits, dogs, pigs, humans, and so forth), and the like, with mammalsbeing of particular interest.

When used as a research tool, a biological sample comprising PKC istypically contacted with a PKC activity-inhibiting amount of a subjectcompound. After the biological sample is exposed to the compound, theeffects of inhibition of a PKC activity are determined usingconventional procedures and equipment, such as the assays disclosedherein. Exposure encompasses contacting the biological sample with thecompound or administering the compound to a subject. The determiningstep can involve measuring a response (a quantitative analysis) or caninvolve making an observation (a qualitative analysis). Measuring aresponse involves, for example, determining the effects of the compoundon the biological sample using conventional procedures and equipment,such as radioligand binding assays and measuring ligand-mediated changesin functional assays. The assay results can be used to determine theactivity level as well as the amount of compound necessary to achievethe desired result, that is, a PKC activity-inhibiting amount.

Additionally, subject compounds can be used as research tools forevaluating other chemical compounds, and thus are also useful inscreening assays to discover, for example, new compounds having a PKCinhibiting activity. In this manner, a subject compound can be used as astandard in an assay to allow comparison of the results obtained with atest compound and with the subject compounds to identify those testcompounds that have about equal or superior activity, if any. Forexample, IC₅₀ data for a test compound or a group of test compounds iscompared to the IC₅₀ data for a subject compound to identify those testcompounds that have the desired properties, for example, test compoundshaving an IC₅₀ about equal or superior to a subject compound, if any.

This aspect includes, as separate embodiments, both the generation ofcomparison data (using the appropriate assays) and the analysis of testdata to identify test compounds of interest. Thus, a test compound canbe evaluated in a biological assay, by a method comprising the steps of:(a) conducting a biological assay with a test compound to provide afirst assay value; (b) conducting the biological assay with a subjectcompound to provide a second assay value; wherein step (a) is conductedeither before, after or concurrently with step (b); and (c) comparingthe first assay value from step (a) with the second assay value fromstep (b). The assays that can be used for generation of comparison dataare disclosed herein, such as the PKC assays.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the embodiments, and are not intended to limit the scope ofwhat the inventors regard as their invention nor are they intended torepresent that the experiments below are all or the only experimentsperformed. Efforts have been made to ensure accuracy with respect tonumbers used (e.g. amounts, temperature, etc.) but some experimentalerrors and deviations should be accounted for. As will be understood, bythose of skill in the art of organic synthesis and medicinal chemistrythe specific conditions set forth below are exemplary and can be variedor adapted to other reagents and products in routine fashion. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used.

As referred to in the Examples, HPLC and LCMS protocols are as follows:

Protocol-1:

-   -   HPLC: Waters 2690 Alliance    -   Diode array detector (210-400 nm)    -   Column: Phenomenex Gemini 4.6×100 mm, 5 μm, 110 Å    -   Column temperature 30° C.    -   Sample temperature 15° C.    -   Solvent A—0.05% Formic acid in Water    -   Solvent B—0.05% Formic acid in Acetonitrile    -   Flow rate—1.5 ml/min

Gradient:

Time A % B % 0 95  5 10 0 100 (curve = 6) 11.1 0 100 11.2 95  5 12.1 95 5

Protocol-2:

-   -   HPLC: Waters 2690 Alliance    -   Diode array detector (210-400 nm)    -   Column: Phenomenex Gemini 4.6×100 mm, 5 μm, 110 Å    -   Column temperature 30° C.    -   Sample temperature 15° C.    -   Solvent A—0.05% Formic acid in Water    -   Solvent B—0.05% Formic acid in Acetonitrile    -   Flow rate—1.5 ml/min

Gradient:

Time A % B % 0 95  5 10 0 100 (curve = 8) 11.1 0 100 11.2 95  5 12.1 95 5

Protocol-3:

-   -   HPLC: Waters 2695 Alliance    -   Diode array detector (210-400 nm)    -   Column: Phenomenex Gemini 4.6×100 mm, 5 μm, 110 Å    -   Column temperature 30° C.    -   Sample temperature 15° C.    -   Solvent A—0.05% Formic acid in Water    -   Solvent B—0.05% Formic acid in Acetonitrile    -   Flow rate—1.5 ml/min

Gradient:

Time A % B % 0 95  5 10 0 100 (curve = 6) 11.1 0 100 11.2 95  5 12.1 95 5

Chiral HPLC Methods:

Protocol-4:

-   -   HPLC: Waters 2695 Alliance    -   Diode array detector (210-400 nm)    -   Column: Chiralcel-OJ, 4.6×250 mm, with guard    -   Mobil phase (isocratic): 40% methanol, 40% ethanol, 19.9%        Hexane, 0.1% triethylamine    -   Flow rate: 0.5 ml/min    -   Injection volume: 3 μL    -   Concentration: approx 5 mg/ml    -   Detection: UV at 254 nm    -   Run Time: 30 minutes

Protocol-5:

-   -   HPLC: Waters 2695 Alliance    -   Diode array detector (210-400 nm)    -   Column: Chiralcel-OJ, 4.6×250 mm, with guard    -   Mobil phase (isocratic): 89.9% Hexane, 5% methanol, 5% ethanol        0.1% triethylamine    -   Flow rate: 0.5 ml/min    -   Injection volume: 3 μL    -   Concentration: approx 5 mg/ml    -   Detection: UV at 254 nm    -   Run Time: 45 minutes

Synthesis of Octahydroindolizine Portions Example 1 Synthesis of(RS,SR,RR,SS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

Preparation of (R/S)-Hexahydro-5,5-dimethylindolizin-7(1H)-one

The compound was prepared as described in J. Chem. Soc., Perkin Trans. 11986, 447-453, which is hereby incorporated by reference in itsentirety.

4-Aminobutyraldehyde diethyl acetal (6.4 g, 39.7 mmol) and mesityl oxide(20 ml, 175.2 mmol) were mixed neat and stirred at room temperature for1 hour. After this time, the reaction mixture was extracted with 2.5 MHCl and the extract washed with ether. The aqueous layer was heated atreflux for 3 hours. The reaction mixture was concentrated in vacuo toreduce the volume by ˜50%. The concentrated aqueous layer was cooled onice and diluted with methylene chloride (˜100 ml). The mixture wasbasified with aqueous potassium hydroxide and the layers separated. Theaqueous layer was further extracted with methylene chloride (100 ml) andthe combined organic extracts were washed with aqueous potassiumcarbonate. The organic layer was dried over potassium carbonate,filtered and concentrated. Purification by flash column chromatography,on silica gel, eluting with neat EtOAc provided the product as paleyellow oil, 3.2 g (48% yield). The mixture can also be purified bydistillation from K₂CO₃ (instead of column chromatography) to afford theketone in a similar yield.

¹H NMR (CDCl₃, 300 MHz) δ: 3.02 (dt, J=8.8, 3.6 Hz, 1H), 2.74-2.84 (m,1H), 2.38-2.54 (m, 3H), 2.09-2.24 (m, 2H), 1.86-2.02 (m, 2H), 1.72-1.82(m, 1H), 1.45-1.57 (m, 1H), 1.24 (s, 3H), 0.92 (s, 3H).

Preparation of (R/S)-Hexahydro-5,5-dimethylindolizin-7(1H)-one-oxime

The compound was prepared as described in J. Chem. Soc., Perkin Trans. 11986, 447-453, which is hereby incorporated by reference in itsentirety.

Mixture of (R,S)-hexahydro-5,5-dimethylindolizin-7(1H)-one-oxime (2.9 g,17.3 mmol) and hydroxylamine hydrochloride (1.2 g, 17.3 mmol) in EtOH(10 ml) and pyridine (10 ml) were heated to reflux and the mixturestirred for 2 hours. A thick precipitate develops. After allowing thereaction mixture to cool to room temperature, the mixture was placed ina −18° C. freezer where it was left for 1 hour. The mixture was thenfiltered, and the filter cake washed with cold EtOH (2×5 ml) to affordthe oxime (3.00 g, 95%) as a solid.

LCMS (m/z): 183 (MH⁺), RT=0.89 min. (Protocol-1)

Preparation of (R/S,S/R,R/R,S/S)-Octahydro-5,5-dimethylindolizin-7-amine

(R,S)-Hexahydro-5,5-dimethylindolizin-7(1H)-one-oxime (3.2 g, 17.6 mmol)was dissolved in AcOH (30 ml). The clear solution is transferred to aPan hydrogenation flask and placed under nitrogen. PtO₂ (0.5 g) is addedto the Parr flask under nitrogen. The mixture was then transferred to aParr hydrogenation apparatus, evacuated and filled with hydrogen (×3).The mixture was hydrogenated at 55-60 psi (optionally topping-uphydrogen) until LC/MS and TLC indicated complete reaction to the amine.After complete reaction, the mixture was placed under nitrogen andfiltered through a small pad of Celite. The filter cake was washed withMeOH (×3) and the filtrate was concentrated under vacuum to leave theoctahydro-5,5-dimethylindolizin-7-amine as a salt—yield assumedquantitative, and amine was used directly in a displacement reactionafter drying under high vacuum.

Preparation of(R/S,S/R,R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

A mixture of octahydro-5,5-dimethylindolizin-7-amine acetic acid salt(4.1 g, 14.1 mmol) and dichloro-5-fluoropyrimidine (2.83 g, 17 mmol) andNaHCO₃ (3.6 g, 42.4 mmol) in MeOH/H₂O (50:10) was stirred at 45° C. for12 hours. Both the diastereomers were detected by LC/MS and TLC.Volatiles were removed and 100 ml of 4 N HCl in dioxane was added to thecrude reaction mixture. Volatiles were removed and the crude mixture wasadsorbed on silica gel. Combi-flash column chromatography was performedto separate the diastereomers using DCM/2N NH₃ in MeOH (90:10) aseluent. Column purification gave the product as a mixture of twodiastereomers [each diastereomer is mixture of two enantiomers] in 74%yield (3.1 g).

A note should be made that the desired isomer can be prepared in a ca.6:1 ratio by use of the Na/pentanol reduction used in J. Chem. Soc.,Perkin Trans. 1 1986, 447-453, which is hereby incorporated by referencein its entirety.

Example 2 Chromatography ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

Preparation of(R/S,S/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amineand (S/S &R/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

A mixture of octahydro-5,5-dimethylindolizin-7-amine acetic acid salt(4.1 g, 14.1 mmol) and dichloro-5-fluoropyrimidine (2.8 g, 17 mmol) andNaHCO₃ (3.6 g, 42.4 mmol) in MeOH/H₂O (50:10) was stirred at 45° C. for12 hours. Both the diastereomers were detected by LC/MS and TLC.Volatiles were removed and 100 ml of 4 N HCl in dioxane was added to thecrude reaction mixture. Volatiles were removed and the crude mixture wasadsorbed on silica gel. Combiflash column chromatography was performedto separate the diastereomers using DCM/2N NH₃ in MeOH (95:5) as eluent.Column purification gave seperated diastereomers D1 & D2 in 12% yield(0.5 g) and 62% yield (2.6 g) respectively as HCl salts.

A small quantity of each diastereomer was neutralized with aq. 1N NaOHand analyzed. Aqueous 1N NaOH was added to diastereomeric HCl salt (50mg) in EtOAc (10 ml) and the layers were separated. Aqueous layer wasworked up twice with EtOAc (10 ml) and the combined organic layers weredried over Na₂SO₄. Removal of the volatiles in vacuo gave the product.

Data for(R/S,S/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(D1): [Single Diastereomer, Mix of Enantiomers]

¹H NMR (CDCl₃, 300 MHz) δ: 7.84 (dd, J=2.8, 1.4 Hz, 1H), 5.02 (br. s,1H), 4.17-4.27 (m, 1H), 2.97 (dt, J=8.5, 2.8 Hz, 1H), 2.57-2.51 (m, 1H),2.38 (q, J=8.5 Hz, 1H), 2.26 (dd, J=11.8, 1.6 Hz, 1H), 1.79-1.90 (m,2H), 1.64-1.78 (m, 2H), 1.33-1.47 (m, 2H), 1.23-1.26 (m, 1H), 1.19 (s,3H), 1.06 (s, 3H).

¹⁹F NMR (DMSO) δ: −159.91.

LCMS (m/z): 299 (MH⁺) (D1 retention time: 4.75 min. see Protocol-2 ingeneral methods).

Data for (S/S,R/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(D2): [Single Diastereomer, Mix of Enantiomers]

¹H NMR (CDCl₃, 300 MHz) δ: 7.86 (d, J=2.8 Hz, 1H), 5.35 (br. d, J=3.6Hz, 1H), 4.37-4.40 (m, 1H), 3.02 (dt, J=8.5, 2.75 Hz, 1H), 2.58-2.50 (m,1H), 2.42 (q, J=8.8 Hz, 1H), 2.05 (dd, J=13.8, 2.2 Hz, 1H), 1.86-1.96(m, 2H), 1.61-1.82 (m, 2H), 1.37-1.61 (m, 3H), 1.18 (s, 3H), 1.08 (s,3H).

LCMS (m/z): 299 (MH⁺) (D2 retention time: 3.99 min. (see Protocol-2 ingeneral methods).

Example 3 Synthesis of(7R,8aS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

Preparation of Preparation of(±)-hexahydro-5,5-dimethylindolizin-7-(1H)-one (Compound 3-3)

This is based on a literature procedure—see F. D. King J. Chem. Soc.,Perkin Trans. 1 1986, 447-453, which is hereby incorporated by referencein its entirety.

4-Aminobutyraldehyde diethyl acetal (Compound 3-1) (94 g, 0.58 mol) andmesityl oxide (Compound 3-2) (290 g, 2.95 mol) were mixed neat andstirred at room temperature for 1 hour. After this time, the reactionmixture was extracted with 2.5 M HCl and the extract washed with ether.The aqueous layer was heated at reflux for 3 hours. The reaction mixturewas concentrated under vacuum to reduce the volume by ˜50%. Theconcentrated aqueous layer was cooled on ice and diluted with methylenechloride (˜300 mL). The mixture was basified with aqueous KOH and thelayers separated. The aqueous layer was further extracted with CH₂Cl₂(200 mL) and the combined organic extracts were washed with aqueousK₂CO₃. The organic layer was dried over K₂CO₃, filtered and concentratedin vacuo to leave a crude residue. The residue was purified by flashcolumn chromatography, on silica gel, eluting with neat methylenechloride to provide the product (45.5 g, 47%) as a pale yellow oil.

¹H NMR (CDCl₃, 300 MHz): δ 2.96 (dt, J=9.0, 3.0 Hz, 1H), 2.79-2.69 (m,1H), 2.48-2.32 (m, 3H), 2.18-2.04 (m, 2H), 1.97-1.63 (m, 3H), 1.51-1.41(m, 1H), 1.18 (s, 3H), 0.87 (s, 3H).

¹³C NMR (CDCl₃, 75 MHz): δ 210.1, 56.8, 54.7, 54.1, 47.3, 44.3, 31.5,30.2, 21.2, 16.6.

It should be noted that, after workup, the reaction mixture can also bepurified by distillation from K₂CO₃ (instead of column chromatography),to afford the ketone product in a similar yield.

Preparation of (±)-octahydro-5,5-dimethylindolizin-7-ol (Compound 3-4)

A solution of 1.0 M L-selectride in THF (419.5 mL, 419.5 mmol) was addeddropwise over ca. 120 minutes to a mixture of(±)-hexahydro-5,5-dimethylindolizin-7-(1H)-one (Compound 3-3) (50.0 g,299 mmol) in anhydrous THF (200 mL) at −78° C. The reaction mixture wasstirred at −78° C. for a further 4 hours. The mixture was lifted fromthe bath and allowed to warm to −15° C. over 1 hour. Analysis of thecrude reaction by TLC indicated complete conversion to alcohol (Compound3-4). The reaction mixture was cooled to −78° C. and quenched by thedropwise addition of MeOH (150 mL). The reaction mixture was thenallowed to warm to room temperature and stirred overnight. The solventwas then removed in vacuo to leave a crude residue. The residue wasdissolved in 3N HCl (200 mL) and extracted with EtOAc (250 mL then 100mL). The aqueous layer was then basified using 12N NaOH to pH 14, andextracted with CH₂Cl₂ (4×500 mL). The combined organic layers were dried(Na₂SO₄), filtered and the solvent removed in vacuo to leave a lightbrown solid. The solid was titurated with Et₂O to give the product (29.2g). The filtrate was concentrated in vacuo and triturated withEt₂O/hexane (1:1) to give a further crop of product (5.8 g). Thecombined yield of the alcohol product (Compound 3-4) was 35.0 g (70%).

¹H NMR (CDCl₃, 300 MHz): δ 4.17 (m, 1H), 2.98 (dt, J=8.7, 3.0 Hz, 1H),2.78-2.68 (m, 1H), 2.43 (q, J=8.7 Hz, 1H), 1.93-1.83 (m, 2H), 1.80-1.54(m, 5H), 1.46-1.34 (m, 2H), 1.16 (s, 3H), 1.14 (s, 3H).

¹³C NMR (CDCl₃, 75 MHz): δ 66.9, 52.5, 50.9, 45.8, 45.1, 39.4, 31.6,31.0, 20.6, 17.1.

An alternative workup using hydrogen peroxide and NaOH is also possible.(±)-Hexahydro-5,5-dimethylindolizin-7-(1H)-one (Compound 3-3) (30.6 g,0.18 mol) was dissolved in anhydrous THF and cooled to −78° C.L-Selectride, 1.0 M in THF (260 mL, 0.26 mol) was added dropwise over 90minutes. The resulting mixture was allowed to stir at −78° C. for 4hours. The reaction was quenched by dropwise addition of hydrogenperoxide, 30% wt in H₂O (150 mL), followed by dropwise addition of 3Nsodium hydroxide solution (150 mL). The cold bath was removed andreplaced with a lukewarm water bath—the resulting mixture was allowed tostir for 1 hour after reaching room temperature. A white precipitateformed and was removed by vacuum filtration through a pad of Celite, andthe filter cake was rinsed with EtOAc (×3). The filtrate was dilutedfurther with EtOAc and H₂O and the layers were separated. The organiclayer was washed with brine, dried (Na₂SO₄), filtered and concentratedin vacuo to provide the product (27.6 g) as a solid. Trituration with anEt₂O/hexane solution (1:1) provided the product (21.5 g, 71% yield) asan off-white solid.

Preparation of (7S,8aS)-octahydro-5,5-dimethylindolizin-7-ol (Compound3-6)

A mixture of (±)-octahydro-5,5-dimethylindolizin-7-ol (Compound 3-4)(20.4 g, 118.3 mmol) and Novozym 435 (20.4 g) in vinyl acetate (400 mL)was slowly stirred (150 revolutions per minute) at room temperature for16 hours. The reaction mixture was then filtered and the filter cakewashed with EtOAc (400 mL). The filtrate was concentrated in vacuo toleave a crude residue that was separated by column chromatography onsilica gel using CH₂Cl₂/2N NH₃ in MeOH (95:5) as eluent to give thedesired chiral alcohol (Compound 3-6) (9.0 g, 44%)

[α]_(D)=−25.1° (c=0.34 in MeOH).

¹H NMR (CDCl₃, 300 MHz): δ 4.19-4.17 (m, 1H), 2.98 (td, J=8.7, 3.0 Hz,1H), 2.78-2.68 (m, 1H), 2.44 (q, J=8.7 Hz, 1H), 1.93-1.82 (m, 2H),1.80-1.57 (m, 5H), 1.47-1.34 (m, 2H), 1.16 (s, 3H), 1.14 (s, 3H).

¹³C NMR (CDCl₃, 75 MHz): δ 66.8, 52.4, 50.8, 45.6, 45.0, 39.2, 31.5,30.8, 20.5, 16.9.

m/z=170.17 (M+H)⁺.

Alternatively, the product can be purified by column chromatography onbasic alumina using EtOAc/hexane (0:1 to 2:3) or CH₂Cl₂/MeOH (1:0 to95:5) as eluent.

The effect of time on the yield and enantiomeric excess from thisresolution is shown below.

Effect upon yield and enantiomeric excess with variation in time forkinetic resolution of (±)-octahydro-5,5-dimethylindolizin-7-ol (Compound3-4)

Absolute yield of recovered Enantiomer excess of alcohol (Compound 3-6)recovered alcohol Time (hours) (theoretical yield) (Compound 3-6) 8 62%48% 16 44% (88% theoretical) >99% 24 40% (80% theoretical) >99% 60 35%(70% theoretical) >99% >72 21% (42% theoretical) >99%

Reactions were undertaken with alcohol 4 (1 equivalent) and Novozym 435(same weight as alcohol) in vinyl acetate at room temperature whilestirring at ca. 150 revolutions per minute.

Enantiomer excess ascertained by measuring enantiomeric excess oftosylate derivative 3-7 by chiral HPLC, after reaction of the alcohol3-6 with p-TsCl, Et₃N and DMAP in CH₂Cl₂.

Other Procedures for Resolution of Racemic Mixture 3-4

Enzymatic Resolution Using Amano Lipase A, from Aspergillus niger

Amano Lipase A, from Aspergillus niger (100 mg; Aldrich catalogue number534781) was added in one portion to a mixture of racemic alcohol (1.0 g,5.9 mmol) in vinyl acetate (20 mL). The mixture was sealed, then stirredat room temperature overnight. TLC analysis indicated no substantialformation of acetate product.

Attempted Enzymatic Resolution Using Amano Lipase a, from Aspergillusniger at 40° C.:

Amano Lipase A, from Aspergillus niger (100 mg; Aldrich catalogue number534781) was added in one portion to a mixture of racemic alcohol (0.5 g,3.0 mmol) in vinyl acetate (10 mL). The mixture was sealed, then heatedto 40° C. and stirred at overnight. TLC analysis indicated no formationof acetate product.

Attempted Enzymatic Resolution Using Amano Lipase M, from Mucorjavanicus

Amano Lipase M, from Mucor javanicus (100 mg; Aldrich catalogue number534803) was added in one portion to a mixture of racemic alcohol (0.5 g,3.0 mmol) in vinyl acetate (10 mL) under nitrogen. The mixture wasstirred at room temperature overnight. TLC analysis indicated noformation of acetate product.

Preparation of(7S,8aS)-octahydro-5,5-dimethylindolizin-7-yl-4-methylbenzenesulfonate(Compound 3-7)

para-Toluenesulfonyl chloride (13.2 g, 69.2 mmol) was added in portionsto a mixture of (7S,8aS)-octahydro-5,5-dimethylindolizin-7-ol (Compound3-6) (9.0 g, 53.25 mmol, 1.0 equiv), 4-N,N-dimethylaminopyridine (9.8 g,79.9 mmol) and Et₃N (11.1 mL, 79.9 mmol) in CH₂Cl₂ (180 mL) at 0° C. Themixture was allowed to warm to room temperature and then stirred for 4days. EtOAc (600 mL) was added, and the resulting solid was filtered andwashed with EtOAc (ca. 150 mL). The filtrate was washed with water (600mL) and concentrated in vacuo to leave a crude residue. The residue waspurified by column chromatography on silica gel using EtOAc/MeOH (9:1)as eluent to give the product (14.5 g, 84%) as a solid.

[α]_(D)=−6.1° (c=0.43 in MeOH).

¹H NMR (CDCl₃, 300 MHz): δ 7.75 (d, J=8.4 Hz, 2H), 7.30 (d, J=8.1 Hz,2H), 4.80-4.78 (m, 1H), 2.92 (td, J=9.0, 3.3 Hz, 1H), 2.69-2.60 (m, 1H),2.41 (s, 3H), 2.39 (q, J=8.7 Hz, 1H), 2.01-1.96 (m, 1H), 1.88-1.78 (m,1H), 1.74-1.68 (m, 2H), 1.65-1.56 (m, 1H), 1.55-1.49 (m, 1H), 1.40-1.26(m, 2H), 1.07 (s, 3H), 1.05 (s, 3H).

¹³C NMR (CDCl₃, 75 MHz): δ 144.5, 134.3, 129.8, 127.6, 78.7, 52.5, 50.9,44.9, 42.9, 36.7, 31.0, 30.6, 21.6, 20.4, 16.6.

m/z=324.42 (M+H)⁺.

Chiral HPLC Conditions:

-   -   Column: Daicel Chemical Industries, Chiralcel OJ, 4.6×250 mm    -   Mobile phase: 1:1 Methanol/Ethanol 0.1% diaethylamine        (isocratic)    -   Flow rate: 0.5 ml/min    -   Run time: 15 minutes    -   Temperature: room temperature    -   Detection: Water 996 PDA    -   HPLC: Waters 2690 Separations Module

Preparation of (7R,8aS)-7-azido-octahydro-5,5-dimethylindolizine(Compound 3-8)

A mixture of(7S,8aS)-octahydro-5,5-dimethylindolizin-7-yl-4-methylbenzenesulfonate(Compound 3-7) (14.5 g, 44.9 mmol) and NaN₃ (8.8 g, 134.7 mmol) in DMF(120 mL) was heated at 80° C. overnight. After cooling, the reactionmixture was diluted with EtOAc (400 mL) and H₂O (300 mL). The aqueousand organic layers were separated and the aqueous layer extracted withEtOAc (200 mL). The combined organic layers were washed with H₂O (200mL), dried (MgSO₄) and concentrated in vacuo to leave the product whichwas directly used in the next step—yield assumed quantitative (8.7 g).

¹H NMR (CDCl₃, 300 MHz): δ 3.48-3.40 (m, 1H), 3.00-2.92 (m, 1H),2.46-2.40 (m, 1H), 2.35 (q, J=8.7 Hz, 1H), 2.01-1.96 (m, 1H), 1.89-1.64(m, 4H), 1.50-1.40 (m, 2H), 1.20 (s, 3H), 0.96 (s, 3H).

m/z=195.08 (M+H)⁺.

Preparation of (7R,8aS)-octahydro-5,5-dimethylindolizin-7-amine(Compound 3-9)

A solution of crude (7R,8aS)-7-azido-octahydro-5,5-dimethylindolizine(Compound 3-8) (assumed 8.7 g) and Pd(OH)₂ (20% weight on carbon; 1.7 g)in MeOH (150 mL) was hydrogenated at 30 psi at room temperature for 6hours. The reaction mixture was filtered through a pad of Celite and thefilter cake was washed with MeOH (200 mL). The filtrate was concentratedunder vacuum to give the product, which was used directly in the nextstep—yield assumed quantitative (7.5 g).

¹H NMR (CDCl₃, 300 MHz): δ 3.03-2.96 (m, 1H), 2.55-2.47 (m, 1H), 2.41(q, J=8.7 Hz, 1H), 2.08-2.03 (m, 1H), 1.93-1.80 (m, 2H), 1.77-1.65 (m,2H), 1.37-1.20 (m, 2H), 1.21 (s, 3H), 1.10-1.06 (m, 1H), 0.99 (s, 3H).

m/z=169.10 (M+H)⁺.

Preparation of(7R,8aS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Compound 3-10)

A mixture of crude (7R,8aS)-octahydro-5,5-dimethylindolizin-7-amine(Compound 3-9) (assumed 7.5 g, 44.9 mmol) and dichlorofluoropyrimidine(7.5 g, 44.9 mmol) in MeOH (120 mL) was stirred at room temperature for20 hours. The solvent was removed in vacuo and the residue was purifiedby column chromatography on silica gel using CH₂Cl₂/2N NH₃ in MeOH(95:5) as eluent to give the product (10.8 g, 80% over 3 steps).

[α]_(D)=−1.7° (c=0.35 in MeOH).

¹H NMR (CDCl₃, 300 MHz): δ 7.89 (d, J=2.7 Hz, 1H), 5.83 (d, J=5.7 Hz,1H), 4.55-4.38 (m, 1H), 3.55-3.45 (m, 1H), 3.25-3.10 (m, 1H), 2.82 (q,J=8.1 Hz, 1H), 2.42-2.38 (m, 1H), 2.32-1.92 (m, 7H), 1.55 (s, 3H), 1.36(s, 3H).

¹³C NMR (CDCl₃, 300 MHz): δ 154.3 (d, J=3.3 Hz), 152.9 (d, J=12.6 Hz),145.1 (d, J=256.1 Hz), 139.7 (d, J=20.3 Hz), 59.0, 58.2, 44.8, 44.0,41.6, 33.9, 28.8, 27.7, 20.0, 17.9.

¹⁹F NMR (CDCl₃, 282 MHz): δ −158.0 (s).

m/z=299.03 (M+H)⁺.

Example 4 Synthesis of 7-Amino-hexahydro-3,3-dimethylindolizin-5(1H)-one

Synthesis of 7-amino-hexahydro-3,3-dimethylindolizin-5 (1H)-one isillustrated in scheme below. Although the absolute stereochemistry wasnot established for any of the intermediates, from the analysis of ¹HNMR spectral pattern indicates that the final product and theintermediates were single diastereomers (racemic). Absolutestereochemistry of individual enantiomers was not established.

Preparation of 5,5-Dimethyl-1-pyrroline

5,5-Dimethyl-1-pyrroline was prepared with reference to Can. J. Chem.1962, 40, 181, which is hereby incorporated by reference in itsentirety.

A suspension of 5,5-dimethyl-1-pyrroline N-oxide (20 g, 177 mmol) andtriphenylphosphine (52.38 g, 200 mmol) were heated at 100° C. for 30minutes, under a short Vigreux column under argon. The liquid whichformed was further heated up to 180° C. under vacuum (20 torr), and thedistillate was collected. The boiling point varied from 46° C. to 58° C.at 20 torr (lit. 104° C. 760 torr) to give 5,5-dimethyl-1-pyrroline(8.60 g, 50%) as a colorless liquid which becomes yellowish uponstanding, even when kept in the refrigerator.

¹H NMR (DMSO d₆, 300 MHz) δ 7.31 (s, 1H), 2.48-2.55 (m, 2H), 1.50-1.55(m, 2H), 1.11-1.15 (m, 6H); m/z=98 (M+H)⁺.

Preparation of 5-Allyl-2, 2-dimethylpyrrolidine

To a solution of 5,5-dimethyl-1-pyrroline (2.3 g, 23.7 mmol) inanhydrous THF (50 ml) 1.0 M solution of allylmagnesium bromide indiethyl ether (43.3 ml, 47.3 mmol) was added dropwise at 0° C. Thereaction mixture was allowed to stir at 0° C. for 1 hour and brought toroom temperature and stirred for 3 hours. Analysis of the reactionmixture by LC-MS indicated the completion of the reaction. The reactionmixture was cooled to 0° C. and quenched with 5 ml of 1N aqueous HCl andpartitioned with 50 ml of EtOAc. Organic layer was separated and theaqueous layer was worked up with 2×50 ml of EtOAc. Combined organiclayers were dried over MgSO₄ and concentrated under vacuum gave theproduct as light yellow oil in 44% yield (1.5 g). The crude product wastaken to the next step without further purification.

LCMS (m/z)=140 (M+H)⁺.

Preparation of 1-(5-Allyl-2,2-dimethylpyrrolidine-1-yl) prop-2-en-1-one

To a solution of 5-allyl-2, 2-dimethylpyrrolidine (1.5 g, 10.5 mmol) in50 ml of anhydrous CH₂Cl₂, triethylamine (3 ml, 21 mmol) and acryloylchloride (0.94 ml, 11.6 mmol) were added at 0° C. The reaction mixturewas stirred at 0° C. for 1 hour and allowed to warm to room temperatureand stirred for overnight. Analysis of the reaction mixture by LCMSindicated the completion of the reaction. Saturated aqueous NaHCO₃ wasadded to the reaction mixture and the layers were separated. Aqueouslayer was worked up twice with CH₂Cl₂ and the combined organic layerswere dried over Na₂SO₄. Removal of the volatiles and purification of thecrude by column chromatography gave the product in 80% yield (1.60 g) ascolorless oil.

¹H NMR (CDCl₃, 300 MHz) δ 6.33-6.52 (m, 2H), 5.68-5.75 (m, 1H), 5.62(dd, J=7.0, 2.6 Hz, 1H), 5.09 (s, 1H), 5.06-5.07 (m, 1H), 3.97-4.00 (m,1H), 2.15-2.33 (m, 2H), 1.88-1.95 (m, 2H), 1.66-1.83 (m, 2H), 1.60 (s,3H), 1.45 (s, 3H); LCMS (m/z)=194 (M+H)⁺.

Preparation of 2,3,8,8a-Tetrahydro-3,3-dimethylindolizin-5-(1H)-one

5-Allyl-2, 2-dimethylpyrrolidine (0.5 g, 2.6 mmol) was taken in a 250 mlRB flask and flushed with nitrogen for three times. To the above flask100 ml of anhydrous CH₂Cl₂ and Grubbs 2^(nd) generation catalyst (0.27g, 0.3 mmol) were added at room temperature. The reaction mixture wasstirred for overnight at room temperature. LCMS analysis indicatedcompletion of the reaction. Volatiles were removed in vacuo and thecrude was purified by column chromatography to give 0.32 g (yield=74%)of the product as colorless oil.

¹H NMR (CDCl₃, 300 MHz) δ 6.38-6.46 (m, appears to be dt, 1H), 5.84 (dd,J=9.7, 3.2 Hz, 1H), 3.69-3.80 (m, 1H), 2.40 (td, J=17.3, 6.2 Hz, 1H),2.07-2.14 (m, 1H), 1.97-2.02 (m, 1H), 1.61-1.84 (m, 3H), 1.56 (s, 3H),1.44 (s, 3H); LCMS (m/z)=166 (M+H)⁺.

Preparation of 7-Azido-3,3-dimethylindolizin-5-(1H)-one

A solution of 2, 3, 8, 8a-tetrahydro-3,3-dimethylindolizin-5(1H)-one(0.3 g, 1.8 mmol) in 10 ml of toluene was taken a 25 ml RB flask. To theabove flask azido-trimethylsilane (2.42 ml, 18.2 mmol), AcOH (1.2 ml, 20mmol) and DBU (0.27 ml, 1.8 mmol) were added. The reaction mixture wasstirred at room temperature for overnight. Volatiles were removed undervacuum and the crude reaction mixture was purified by columnchromatography. The product was obtained as colorless oil in 85% yield(0.32 g).

¹H NMR (CDCl₃, 300 MHz) δ 4.10-4.13 (m, 1H), 3.68-3.78 (m, 1H), 2.58(dd, J=17.9, 5.6 Hz, 1H), 2.42-2.49 (m, 1H), 2.14-2.20 (m, 1H),1.90-1.96 (m, 1H), 1.72-1.80 (m, 2H), 1.55 (s, 3H), 1.47-1.49 (m, 2H),1.42 (s, 3H); LCMS (m/z)=209 (M+H)⁺.

The product from the above reaction appears to be a singlediastereomer—see Journal of Organic Chemistry, 71, 6630-6633; 2006,which is hereby incorporated by reference in its entirety, for a similaraddition of TMS-N₃ to a tetrahydroindolizin-5(1H)-one system to give asingle diastereomeric product.

Preparation of 7-amino-hexahydro-3,3-dimethylindolizin-5 (1H)-one

7-Azido-3,3-dimethylindolizin-5 (1H)-one (0.3 g, 1.5 mmol) is dissolvedin EtOH (20 ml). The clear solution is transferred to a Parrhydrogenation flask and placed under nitrogen. 10% Pd-C(0.25 g) wasadded to the Parr flask under nitrogen. The mixture was then transferredto a Pan hydrogenation apparatus, evacuated and filled with hydrogen(×3). The mixture was hydrogenated at 30 psi (optionally topping-uphydrogen) until LC/MS and TLC indicated complete reaction to the amine.After complete reaction, the mixture was placed under nitrogen andfiltered through a small pad of Celite. The filter cake was washed withMeOH (×3) and the filtrate was concentrated under vacuum to leave the7-amino-hexahydro-3, 3-dimethylindolizin-5-(1H)-one in 89% yield (0.25g).

¹H NMR (CDCl₃, 300 MHz) δ 3.75-3.92 (m, 1H), 3.50-3.54 (m, 1H), 2.54(dd, J=16.9, 5.9 Hz, 1H), 2.13 (dd, J=17.6, 1.8 Hz, 1H), 1.87-1.95 (m,2H), 1.67-1.76 (m, 2H), 1.56 (s, 3H), 1.44-1.53 (m, 2H), 1.42 (s, 3H);LCMS (m/z)=183 (M+H)⁺.

Example 5 Synthesis of(R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amineand (R,S/S, R)—N-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amine

Preparation of (R/S)-octahydroindolizin-7-one

The compound was prepared according to J. Chem. Soc., Perkin Trans. 11986, 447-453, which is hereby incorporated by reference in itsentirety.

A mixture of but-3-en-2-one (5.4 g, 75 mmol) was added dropwise over ca.10 minutes to a solution of 4,4-diethoxybutan-1-amine (9.6 g, 60 mmol)in Et₂O (30 ml) at 0° C. under nitrogen. The mixture was stirred at 0°C. for a further 60 minutes then extracted with 2.5 M HCl (150 ml). Theaqueous acid layer was then heated to reflux and stirred for 150minutes. After allowing to cool to room temperature, the mixture wasconcentrated in vacuo to about one third of the original volume. Themixture was cooled to 0° C. and CH₂Cl₂ (200 ml) was added, followed by3N NaOH until pH>10. The aqueous and organic layers were partitioned andthe organic layer was washed with a K₂CO₃ solution (×1). The combinedaqueous layers were extracted with CH₂Cl₂ (2×150 ml) and then thecombined organic layers were dried (K₂CO₃), filtered and concentrated invacuo. The residue was dry-loaded on to silica gel and purified bycolumn chromatography on silica gel using CH₂Cl₂/MeOH (100:0 to 94:6 inincrements of 2% MeOH) to give the desired product (2.0 g, 24%) as anoil.

¹H NMR (300 MHz; CDCl₃) δ 3.37-3.30 (m, 1H), 3.20-3.14 (m, 1H),2.68-2.51 (m, 2H), 2.40-2.19 (m, 5H), 2.03-1.93 (m, 2H), 1.91-1.80 (m,1H), 1.60-1.48 (m, 1H).

¹³C NMR (75 MHz; CDCl₃) δ 209.3, 64.2, 53.3, 50.3, 47.4, 40.7, 31.5,22.6.

Preparation of (R/S)-octahydroindolizin-7-one oxime

The compound was prepared according to J. Chem. Soc., Perkin Trans. 11986, 447-453, which is hereby incorporated by reference in itsentirety.

A mixture of octahydroindolizin-7-one (1.8 g, 12.9 mmol) andhydroxylamine hydrochloride (0.8 g, 12.9 mmol) in EtOH (10 ml) andpyridine (10 ml) was heated to reflux and stirred for 90 minutes. Afterallowing to cool, the mixture was concentrated in vacuo to leave a crudesolid (a bath temperature of 50° C. was used to remove solvent). Thesolid was triturated with cold (−18° C.) EtOH and filtered and thefilter cake was washed with a further small portion of cold (−18° C.)EtOH. The solid was used directly in the next step and the yield wasassumed quantitative=2.0 g. m/z=155.01 (M+H)⁺; rt=0.90 min (HPLCprotocol-1).

Preparation of (R/R,R/S,S/R,S/S)-octahydroindolizin-7-amine

A mixture of octahydroindolizin-7-one oxime (2.0 g, 12.9 mmol) andplatinum(IV) oxide (0.5 g) in AcOH (30 ml) was hydrogenated at 60 psiovernight. The mixture was filtered through Celite and the filter cakewashed with EtOH (3×30 ml). The filtrate was concentrated in vacuo togive the product as an acetate salt. The yield was assumed to bequantitative=1.81 g of the free amine.

Synthesis of(R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amineand (R,S/S, R)—N-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amine

A mixture of 2,4-dichloro-5-fluoropyrimidine (3.45 g, 20.7 mmol),(R/R,R/S,S/R,S/S)-octahydroindolizin-7-amine (1.81 g, 12.9 mmol) andNaHCO₃ (2.71 g, 32.3 mmol) in MeOH (66 ml) and H₂O (22 ml) was heated at55° C. and stirred for 5 hours. After cooling, the MeOH was removed invacuo. CH₂Cl₂ (100 ml) and H₂O (100 ml) was added to the residue and theorganic and aqueous layers were partitioned. The aqueous layer wasextracted with CH₂Cl₂ (2×50 ml) and the combined organic layers weredried (MgSO₄), filtered and the solvent removed in vacuo to leave acrude oil. The crude residue was dry-loaded on to silica gel andpurified by column chromatography on silica gel (ISCO Redisep Rf Goldcolumn) using a 2M NH₃ in MeOH/CH₂Cl₂ gradient system to give a purefirst-eluting diastereomer (small quantity), mixed fractions, asecond-eluting pure diastereomer and mixed fractions.

Data for second-eluting pure diastereomer: ¹H NMR (300 MHz; CDCl₃) δ7.86 (t, J=2.6 Hz, 1H), 5.33 (br. d, J=7.4 Hz, 1H), 4.20-4.07 (m, 1H),3.25-3.12 (m, 2H), 2.32-2.10 (m, 5H), 1.99-1.46 (m, 5H), 1.30 (q, J=11.5Hz, 1H); ¹³C NMR (75 MHz; CDCl₃) δ 154.6 (d, J=4.4 Hz), 153.1 (d, J=12.0Hz), 143.4 (d, J=254.8 Hz), 139.5 (d, J=20.1 Hz), 63.3, 53.4, 50.4,48.4, 37.0, 31.6, 30.1, 21.5; ¹⁹F NMR (282 MHz; CDCl₃) δ −159.4;m/z=271.08 (M+H)⁺ for ³⁵Cl; rt=1.91 min (HPLC protocol-1).

The mixed fractions from the first column were combined and re-columnedusing the same silica gel and gradient system to give a purefirst-eluting diastereomer, a pure second-eluting diastereomer and mixedfractions.

Data for pure first-eluting diastereomer from the above column:m/z=271.04 (M+H)⁺ for ³⁵Cl; rt=2.35 min (HPLC protocol-1).

Data for pure second-eluting diastereomer from the above column:m/z=269.05 (M−H)⁺ for ³⁵Cl; rt=1.97 min (HPLC protocol-1).

Example 6 Synthesis of(±)-2-chloro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carboxamide

A mixture of (±)-octahydro-5,5-dimethylindolizin-7-amine (0.65 g, 3.9mmol) in MeOH (10 ml) was added to a stirred solution of2,4-dichloropyrimidine-5-carboxamide (0.75 g, 3.9 mmol; preparedaccording to the procedure set forth in US patent applicationpublication US20110130415, page 41) in MeOH (30 ml) and H₂O (4 ml) at 0°C. under nitrogen. After complete addition, the mixture was slowlywarmed to room temperature and stirred at room temperature overnight.The mixture was then concentrated in vacuo and the residue partitionedbetween EtOAc (150 ml) and 1N NaOH (100 ml). The aqueous layer wasextracted with EtOAc (1×100 ml) and the combined organic extracts weredried (Na₂SO₄), filtered and the solvent removed in vacuo. The residuewas dry-loaded on to basic alumina (Brockmann grade IV) and thenpurified by column chromatography on basic alumina (Brockmann grade IV)using CH₂Cl₂/MeOH (1:0 to 95:5) as eluent to give the product (550 mg).

Note: the product eluted very quickly from the basic alumina column, anda by-product also co-eluted with the desired compound. This by-productis believed to be(±)-2-methoxy-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carboxamide.However, the mixture (550 mg) was used directly in the next step withoutfurther purification.

Example 7 Synthesis of(±)-2-chloro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carbonitrile

Trifluoroacetic anhydride (460 μL, 3.24 mmol) was added dropwise over5-10 minutes to a stirred mixture of(±)-2-chloro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carboxamide(500 mg; contaminated with(±)-2-methoxy-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carboxamide)and Et₃N (970 μL, 7.0 mmol) in THF (15 ml) at −40° C. (internaltemperature) under nitrogen. The mixture was stirred at −40 to −50° C.(internal temperature) for 30 minutes, then allowed to warm to −30° C.(internal temperature) and the mixture stirred for 15 minutes. TLCanalysis indicated completion of the reaction, so the mixture wasworked-up by pouring in to an ice-H₂O mixture (50 ml) and EtOAc (50 ml).The aqueous and organic layers were partitioned and the aqueous layerwas extracted with EtOAc (1×50 ml). The combined organic extracts weredried (MgSO₄), filtered and the solvent removed in vacuo to leave acrude residue. The residue was dry-loaded on to basic alumina (Brockmanngrade IV) and purified by column chromatography on basic alumina(Redisep 24 g basic alumina column) using CH₂Cl₂/MeOH (gradient from 1:0to 93:7) as eluent to give the product (128 mg). LC/MS indicated theproduct to be of about 70% purity and it was used directly in the nextstep (the contaminant wasN-(5-cyano-2-methoxypyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine).m/z=306.13 (M+H)⁺ for ³⁵Cl; rt=2.64 min (HPLC protocol-1).

Also obtained from the column was a sample containing mostlyN-(5-cyano-2-methoxypyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(100 mg). This was purified by high-performance liquid chromatography inorder to confirm identity. m/z=302.17 (M+H)⁺; rt=2.87 min (HPLCprotocol-1).

Example 8 Synthesis of7-(2-Chloro-5-fluoropyrimidin-4-ylamino)-hexahydro-5,5-dimethylindolizin-3(5H)-one

Preparation of Methyl 3-(6,6-dimethyl-4-oxopiperidin-2-yl)propanoate

Diacetoneamine hydrogen oxalate (4.25 g) and methyl 4-oxobutanoate (2 g)were suspended in methanol (20 ml). The reaction mixture were stirred at100° C. for three hours and then at room temperature overnight. It wasthen evaporated and the residue was purified by Combiflashchromatography (2.0 M ammonia methanol in dichloromethane=0-30%) to givemethyl 3-(6,6-dimethyl-4-oxopiperidin-2-yl)propanoate (1.35 g, 37%).

¹H NMR (300 MHz; CDCl₃) δ 3.62 (s, 3H), 3.21-3.16 (m, 1H), 2.41-2.32 (m,4H), 2.20 (dd, J=1.8, 13.8 Hz, 2H), 1.85 (p, J=6.6 Hz, 2H), 1.28 (s,3H), 1.09 (s, 3H).

Preparation of Hexahydro-5,5-dimethylindolizine-3,7-dione

Methyl 3-(6,6-dimethyl-4-oxopiperidin-2-yl)propanoate (1.35 g) wasdissolved in methanol (25 ml) and 1.0 N sodium hydroxide aqueoussolution was added (7.6 ml, 1.2 eq.). The reaction mixture was stirredat room temperature overnight. It was then evaporated and pumped todryness to give the acid.

The acid was dissolved in THF (20 ml) and dichloromethane (100 ml). Tothis solution, was added thionyl chloride (0.69 ml, 1.5 eq.) dropwise.It was then stirred at 40° C. for three hours and evaporated. Theresidue was purified by Combiflash chromatography (2.0 M ammoniamethanol in dichloromethane=0-30%) to givehexahydro-5,5-dimethylindolizine-3,7-dione.

¹H NMR (300 MHz; d₆-DMSO) δ 3.24 (m, 1H), 2.59 (dd, J=3.6, 18.0 Hz, 1H),2.34-2.10 (m, 5H), 1.54-1.47 (m, 2H), 1.41 (s, 3H), 1.32 (s, 3H).

Preparation of7-(2-Chloro-5-fluoropyrimidin-4-ylamino)-hexahydro-5,5-dimethylindolizin-3(5H)-one

Hexahydro-5,5-dimethylindolizine-3,7-dione (230 mg) and ammonium acetate(990 mg, 10 eq.) were dissolved in methanol (5 ml). To the reactionmixture, was added sodium cyanoborohydride (56 mg, 0.7 eq.). Thereaction mixture was stirred at room temperature overnight. LC-MS showedthe formation of the amine. The reaction mixture was quenched with 10 N.HCl aqueous solution to pH 2. Then it was stirred for two hours.

To the reaction mixture, was added sodium carbonate to pH 7. Then2,4-dichloro-5-fluoropyrimidine (500 mg) was added. It was then stirredat room temperature for three days and evaporated. The residue waspurified by Combiflash chromatography (2.0 M ammonia methanol indichloromethane=0-30%) to give two isomers of7-(2-chloro-5-fluoropyrimidin-4-ylamino)-hexahydro-5,5-dimethylindolizin-3(5H)-one(a: 55 mg; b. 110 mg).

Example 9 Synthesis ofN-(2-Chloro-5-fluoropyrimidin-4-yl)-2,2-difluoro-5,5-dimethyloctahydroindolizin-7-amine

Preparation of (S)-tert-butyl4,4-difluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylate

To a THF (30 ml) solution of(S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidine-2-carboxylic acid(3.77 g, 15 mmol) at 0° C., BH₃.THF solution (1.0 M in THF, 45 ml) wasadded dropwise over 20 minutes. The reaction mixture was allowed to warmto room temperature and stirring was continued for a total of 16 hours.Reaction went to completion as monitored by LC-MS. The reaction mixturewas quenched by the addition of saturated aqueous solution of NaHCO₃ (60ml) at room temperature and the stirring was continued at roomtemperature for 4 hours. Most THF was removed in vacuo and the mixturewas extracted with EtOAc (˜60 ml), two layers were separated, organiclayer was washed with brine (˜50 ml), repeat the extraction/wash cycleone more time. Organic layers were combined, dried (Na₂SO₄), filtered,solvent was removed in vacuo. Crude product was purified by silica gelchromatography (Hexane/EtOAc, gradient from 100:0 to 50:50). Compound(S)-tert-butyl 4,4-difluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylatewas obtained as a colorless oil: 2.6473 g (74% yield).

¹H NMR (300 MHz, CDCl₃) δ 4.18 (br s, 1H), 3.88-3.58 (m, 4H), 2.58-2.41(m, 1H), 2.15 (br s, 1H), 1.59-1.33 (m, 9H, overlapped with water peak);LRMS (M+H—“Boc”) m/z 137.96.

Preparation of (R)-tert-Butyl2-(cyanomethyl)-4,4-difluoropyrrolidine-1-carboxylate

To a CH₂Cl₂ (22 ml) solution of (S)-tert-butyl4,4-difluoro-2-(hydroxymethyl)pyrrolidine-1-carboxylate (2.65 g, 11.2mmol) and Et₃N (2.3 ml, 16.8 mmol), at 0° C., methylsulfonyl chloride(0.95 ml, 12.3 mmol) was added dropwise over ˜2 minutes, ice bath wasremoved after 15 minutes, and stirring was continued at room temperaturefor another 30 minutes. Reaction went to completion as monitored byLC-MS. The reaction mixture was cooled to 0° C. and was quenched byaddition of saturated aqueous solution of NaHCO₃ (˜30 ml), stifling wascontinued at 0° C. for 5 minutes and at room temperature for 15 minutes.Two layers were separated, aqueous layer was extracted with CH₂Cl₂ (˜30ml). Organic layers were combined, washed with brine (˜25 ml), dried(Na₂SO₄), filtered, solvent was removed in vacuo. Compound((S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)methylmethanesulfonate was obtained as a very light yellow oil: 3.40 g (96%crude yield).

¹H NMR (300 MHz, CDCl₃) δ 4.42-4.23 (m, 3H), 3.84 (br s, 1H), 3.64(dddd, J=12.6, 12.6, 12.6, 0.9 Hz, 1H), 3.04 (s, 3H), 2.59-2.43 (m, 2H),1.49 (s, 9H); LRMS (M+H—“Boc”) m/z 215.97. Crude product was useddirectly in next reaction without further purification.

A DMSO (40 ml) solution of((S)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)methylmethanesulfonate and NaCN (1.59 g, 3.24 mmol) was stirred at 45° C. for4 hours and at 50° C. for 2 days, the progress of the reaction wasmonitored by LC-MS. After cooling to room temperature, the reaction wasquenched by the addition of water (˜60 ml). Reaction mixture wasextracted with EtOAc (˜40 ml), two layers were separated, organic layerwas washed with brine (˜25 ml), the extraction and wash sequence wasrepeated for two more times. Organic layers were combined, dried(Na₂SO₄), filtered, solvent was removed in vacuo. Product was purifiedby silica gel chromatography (Hexane/EtOAc, gradient from 100:0 to80:20). Compound (R)-tert-butyl2-(cyanomethyl)-4,4-difluoropyrrolidine-1-carboxylate was obtained as acolorless oil: 2.10 g (76% yield over 2 steps).

¹H NMR (300 MHz, CDCl₃) δ 4.31-4.22 (m, 1H), 3.86-3.63 (m, 2H),2.88-2.56 (m, 3H), 2.49-2.34 (m, 1H), 1.48 (s, 9H); LRMS (M+H—“Boc”) m/z146.94.

Preparation of (R)-tert-Butyl2-((N-methoxy-N-methylcarbamoyl)methyl)-4,4-difluoropyrrolidine-1-carboxylate

A MeOH (20 ml) solution of (R)-tert-butyl2-(cyanomethyl)-4,4-difluoropyrrolidine-1-carboxylate (2.10 g, 8.5 mmol)and 0.5 N NaOH aqueous solution (34 ml, 17.0 mmol) was heated at 70° C.for 6 days until desired product became major, as monitored by LC-MS.Significant amount of de-Boc product was also observed. After cooling toroom temperature, MeOH was removed in vacuo, aqueous mixture wasextracted with Et₂O (˜30 ml) which was discarded (note: mainlycarboxamide intermediate). Aqueous layer was acidified with 6N HCl (aq.)until pH≦2 and was extracted with EtOAc (˜25 ml×3). Organic layers werecombined, dried (Na₂SO₄), filtered, solvent was removed in vacuo. Alight brown oil was obtained: 1.62 g; LRMS (M+H—“Boc”) m/z 166.89.

Additional product was recovered from aqueous layer by adding Boc grouponto de-Boc by-product as follows: aqueous layer was basified to pH 8 bythe addition of solid NaHCO₃, and was concentrated to ˜15 ml in volume;to this aqueous mixture, THF (20 ml) was added, followed by Boc₂O (1.2g, 5 mmol). The mixture was stirred at room temperature for 1 hour,LC-MS indicated the complete formation of the product. The reactionmixture was worked-up as above described and 0.4 g of product wasobtained.

To a CH₂Cl₂ (10 ml) solution of2-((R)-1-(tert-butoxycarbonyl)-4,4-difluoropyrrolidin-2-yl)acetic acidand hydroxylamine hydrogen chloride (713.8 mg, 7.32 mmol), EDCI.HCl(1.64 g, 8.54 mmol) and NMM (1.5 ml, 13.4 mmol) were added, the reactionmixture was stirred at room temperature for 15 hours. The reaction wentto completion monitored by LC-MS and was quenched by addition ofsaturated aqueous NaHCO₃ solution (˜25 ml). Two layers were separated,aqueous layer was extracted with CH₂Cl₂ (˜20 ml). Organic layers werecombined, dried (Na₂SO₄), filtered, solvent was removed in vacuo. Crudeproduct was purified by silica gel chromatography (Hexane/EtOAc,gradient from 100:0 to 70:30). Compound (R)-tert-butyl2-((N-methoxy-N-methylcarbamoyl)methyl)-4,4-difluoropyrrolidine-1-carboxylatewas obtained as a light yellow oil: 1.20 g; additional product wasobtained from recovered carboxylic acid: 0.2 g (53% combined overallyield over 2 steps).

¹H NMR (300 MHz, CDCl₃) δ 4.46-4.40 (m, 1H), 3.80-3.58 (m, 1H,overlapped), 3.69 (s, 3H, overlapped), 3.17 (s, 3H), 3.10-2.93 (m, 2H),2.73-2.53 (m, 2H), 2.38-2.26 (m, 1H), 1.47 (s, 9H); LRMS (M+H—“Boc”) m/z208.98.

Preparation of (R)-tert-Butyl4,4-difluoro-2-(4-methyl-2-oxopent-3-enyl)pyrrolidine-1-carboxylate

To a THF (23 ml) solution of (R)-tert-butyl2-((N-methoxy-N-methylcarbamoyl)methyl)-4,4-difluoropyrrolidine-1-carboxylate(1.4 g, 4.54 mmol) over ice bath, 2-methyl-1-propenylmagnesium bromidesolution (0.5 M in THF, 45 ml, 22.7 mmol) was added dropwise over 1hour. Remove ice bath, stifling was continued for another 3 hours. Thereaction was quenched with 1N HCl (aq., 30 ml) at 0° C., and was stirredat room temperature for 30 minutes. Two layers were separated, aqueouslayer was extracted with EtOAc (25 ml×2), organic layers were combined,dried (Na₂SO₄), filtered, solvent was removed in vacuo. Product waspurified by silica gel chromatography (Hexane/EtOAc, linear gradientfrom 100:0 to 80:20). Compound (R)-tert-butyl4,4-difluoro-2-(4-methyl-2-oxopent-3-enyl)pyrrolidine-1-carboxylate wasobtained as a light yellow oil: 1.10 g (79.5% yield).

¹H NMR (300 MHz, CDCl₃) δ 6.04 (s, 1H), 4.41-4.35 (m, 1H), 3.75-3.58 (m,2H), 2.69-2.51 (m, 2H), 2.27-2.14 (m, 1H), 2.14 (s, 3H), 1.89 (s, 3H),1.74 (dt, J=19.4, 10.4 Hz, 1H), 1.46 (s, 9H); LRMS (M+H—“Boc”) m/z204.07.

Preparation of(R)-2,2-Difluoro-hexahydro-5,5-dimethylindolizin-7(1H)-one

A HCO₂H (20 ml) solution of (R)-tert-butyl4,4-difluoro-2-(4-methyl-2-oxopent-3-enyl)pyrrolidine-1-carboxylate(1.10 g, 3.6 mmol) was stirred at room temperature for 6 hours untilonly trace amount of SM was detected by LC-MS. Solvent was removed invacuo with bath temperature ≦23° C. Compound1-((R)-4,4-difluoropyrrolidin-2-yl)-4-methylpent-3-en-2-one was obtainedas a light brown oil and was used directly in next reaction.

¹H NMR (300 MHz, CDCl₃) δ 8.08 (s, >1H, HCO₂H), 7.39 (br s, >2H, H⁺),6.06-6.05 (m, 1H), 4.14-4.04 (m, 1H), 3.68 (dd, J=24.5, 13.2 Hz, 1H),3.54 (td, J=13.9, 10.4 Hz, 1H), 3.09 (dd, J=18.2, 7.4 Hz, 1H), 2.91 (dd,J=18.2, 5.2 Hz, 1H), 2.68-2.55 (m, 1H), 2.42-2.23 (m, 1H), 2.16 (d,J=1.0 Hz, 3H), 1.93 (d, J=1.0 Hz, 3H); LRMS (M+H) m/z 204.08.

A MeOH (350 ml) solution of1-((R)-4,4-difluoropyrrolidin-2-yl)-4-methylpent-3-en-2-one and K₂CO₃(2.5 g, 18 mmol) was stirred at 40° C. for 15 hours, the reaction wentto completion as monitored by LC-MS. Solvent was removed in vacuo,remaining material was suspended in CH₂Cl₂, solid was filtered off,filtrate was collected and solvent was removed in vacuo. Product waspurified by silica gel chromatography (Hexane/EtOAc, linear gradientfrom 100:0 to 80:20). Compound(R)-2,2-difluoro-hexahydro-5,5-dimethylindolizin-7(1H)-one was obtainedas a light yellow solid: 637.4 mg (69% yield over 3 steps).

¹H NMR (300 MHz, CDCl₃) δ 3.39 (ddd, J=14.2, 10.8, 7.6 Hz, 1H),3.16-3.06 (m, 1H), 2.87 (ddd, J=19.1, 14.5, 10.8 Hz, 1H), 2.51-2.39 (m,3H), 2.32 (dd, J=22.4, 11.0 Hz, 1H), 2.20 (dd, J=13.6, 2.1 Hz, 1H),2.15-1.96 (m, 1H), 1.23 (s, 3H), 0.95 (s, 3H); LRMS (M+H) m/z 204.33.

Preparation of(8aR)-2,2-Difluoro-octahydro-5,5-dimethylindolizin-7-amine

A toluene (12 ml) solution of(R)-2,2-difluoro-hexahydro-5,5-dimethylindolizin-7(1H)-one (487.8 mg,2.4 mmol) and (S)-1-phenylethylamine (306 μL, 2.4 mmol) was heated underreflux in a flask equipped with a Dean-Stark distillation receiver.After 22 hours, most solvent was distilled off through Dean-Starkdistillation receiver, and remaining solvent was removed in vacuo.Compound(1S)—N—((R)-2,2-difluoro-hexahydro-5,5-dimethylindolizin-7(1H)-ylidene)-1-phenylethanaminewas obtained as a brown oil and was used directly in next reaction.

Over an ice bath, to a MeOH (10 ml) solution of(1S)—N—((R)-2,2-difluoro-hexahydro-5,5-dimethylindolizin-7(1H)-ylidene)-1-phenylethanamine,NaBH₄ (136 mg, 3.6 mmol) was added. After 30 minutes, reaction went tocompletion monitored by LC-MS. The reaction was quenched by the additionof saturated aqueous NaHCO₃ (10 ml), and most MeOH was removed in vacuo.More water (˜10 ml) was added and aqueous solution was extracted withEtOAc (15 ml×3). Organic layers were combined, dried (Na₂SO₄), filtered,solvent was removed in vacuo. A light brown oil was obtained (690 mg:LRMS (M+H) ink 309.22) and was dissolved in MeOH (5 ml). This MeOHsolution was cooled with ice bath, and concentrated HCl aqueous solution(0.3 ml) was added and mixed well. Volatiles was removed in vacuo andprovided a brownish-green oil: 869 mg as HCl salt and was used directlyin next reaction.

A MeOH (20 ml) solution of(8aR)-2,2-difluoro-octahydro-5,5-dimethyl-N—((S)-1-phenylethyl)indolizin-7-aminehydrogen chloride and Pd(OH)₂—C(20% on activated carbon, 50% wet, 0.2 g)was hydrogenated in a Parr flask under 45 psi of hydrogen. AdditionalPd(OH)₂—C was added (˜0.2 g) was added at 16 hours and 24 hours, and thereaction went to completion at 39 hours monitored by LC-MS. Solid wasremoved by filtration through a short Celite column, washing with MeOH.Filtrate was collected, solvent was removed in vacuo. Compound(8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-amine hydrogenchloride was obtained as an off-white solid and was used directly innext reaction: 0.56 g; LRMS] (M+H) m/z 204.99.

Preparation ofN-(2-Chloro-5-fluoropyrimidin-4-yl)-2,2-difluoro-5,5-dimethyloctahydroindolizin-7-amine

To a MeOH-H₂O (4:1, 10 ml) solution of(8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-amine hydrogenchloride (crude product from above reaction, ˜2.4 mmol) and5-fluoro-2,4-dicholopyrimidine (480.9 mg, 2.88 mmol) was stirred at 30°C. for 25 hours until only trace amount of amine starting material wasdetected by LC-MS. MeOH was removed in vacuo. Additional H₂O (˜10 ml)was added and the mixture was extracted with EtOAc (10 ml×3). Organiclayers were combined, dried (Na₂SO₄), filtered, solvent was removed invacuo. Two sets of diastereomers were observed with ratio of 3.1:1(retention time of 5.6 min and 6.7 min under analytical HPLC conditionsused: C18 column, solvents H₂O with 0.05% HCOOH and Acetonitrile with0.05% HCOOH, a linear gradient from 90:10 to 0:100 over 9 minutes with aflow rate of 1.2 ml/min). Some level of diastereomer separation wasachieved by prep-TLC (CH₂Cl₂:7N NH₃ in MeOH=97:3) and silica gelchromatography (CH₂Cl₂/2N NH₃ in MeOH, linear gradient from 100:0 to95:5), pure Diastereomer 1(7S,8aR)—N-(2-chloro-5-fluoropyrimidin-4-yl)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-aminewas obtained as an off-white solid: ˜300 mg; [α]_(D) ²⁰ −59.4, c=0.96 inMeOH.

¹H NMR (300 MHz, CDCl₃) δ 7.90 (d, J=2.7 Hz, 1H), 5.30 (br s, 1H),4.48-4.36 (m, 1H), 3.35 (ddd, J=13.8, 10.7, 7.4 Hz, 1H), 3.00-2.90 (m,1H), 2.82 (ddd, J=19.5, 15.1, 10.7 Hz, 1H), 2.43-2.29 (m, 1H), 2.10(ddd, J=13.7, 4.5, 2.4 Hz, 1H), 2.02-1.77 (m, 3H), 1.66 (ddd, J=13.8,11.8, 4.3 Hz, 1H), 1.15 (s, 3H), 1.12 (s, 3H); LRMS] (M+H) m/z 335.13.

Diastereomer 2(7R,8aR)—N-(2-chloro-5-fluoropyrimidin-4-yl)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-aminewas obtained as an off-white solid: 100 mg; ¹H NMR (300 MHz, CDCl₃) δ7.88 (d, J=2.8 Hz, 1H), 4.93 (br d, J=8.0 Hz, 1H), 4.31-4.18 (m, 1H),3.30 (ddd, J=14.4, 10.7, 6.6 Hz, 1H), 2.98-2.89 (m, 1H), 2.77 (ddd,J=19.2, 15.3, 10.7 Hz, 1H), 2.43-2.27 (m, 2H), 2.05-1.82 (m, 2H), 1.42(dd, J=12.2, 12.2 Hz, 1H), 1.22-1.14 (m, 1H, overlapped), 1.16 (s, 3H,overlapped), 1.08 (s, 3H); LRMS (M+H) ink 335.13.

Relative stereochemistry of Diastereomer-1 and Diastereomer-2 wasassigned by using 1D-NOE technique after the assignment of relevantprotons via COSY experiment: for Diastereomer-2, positive NOE wasobserved between H7 and H8a, H7 and CH₃ which was absent inDiastereomer-1.

Example 10 Synthesis ofN-(2-Chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5,8-trimethylindolizin-7-amine

Preparation of Hexahydro-5,5,8-trimethylindolizin-7(1H)-one

Hexahydro-5,5-dimethylindolizin-7(1H)-one (racemic) (1.41 g, 8.43 mmol)was dissolved in dry THF (50 ml); the flask was flushed with nitrogenand cooled to −78° C. LDA (5.9 ml, 11.8 mmol) was added via syringe andstirred for 30 minutes. Subsequently, neat methyl iodide (1.30 ml, 21.1mmol) was added via syringe. The clear, pale yellow solution was stirredovernight and allowed to warm to room temperature. The reaction was thenquenched with a saturated NH₄Cl solution and extracted with ethylacetate (3×40 ml). The organic phases were passed through a plug ofMgSO₄ and solvents were evaporated in vacuo. The crude product (849 mg)was obtained in form of a yellow-brown oil and used without furtherpurification in the next step.

Preparation of Octahydro-5,5,8-trimethylindolizin-7-amine

The crude product from the above reaction was dissolved in a 7Mmethanolic NH₃ (20 ml) solution and stirred for 6 hours at roomtemperature. NaBH₄ (849 mg, 12.6 mmol) was suspended in dry THF in aseparate flask and cooled to −45° C. using a dry-ice/MeCN bath. Thesolution containing the imine was then transferred by cannulation intothe flask with the reducing agent. The reaction mixture was stirred for1 hour at −45° C. and then allowed to warm to room temperatureovernight. Water (1 ml) was added to quench remaining hydride thensolvents were removed in vacuo. The reaction mixture was passed througha plug of MgSO₄ to remove water and salts. After evaporation of solventsthe remaining crude product (829 mg) was used without furtherpurification in the next reaction.

Preparation ofN-(2-Chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5,8-trimethylindolizin-7-amine

The product from the above reaction (829 mg, 4.55 mmol) was combinedwith 2,4-dichloro-5-fluoro-pyrimidine (911 mg, 5.45 mmol) and NaHCO₃(457 mg, 5.45 mmol). A 4:1 mixture of MeOH:H₂O (50 ml) was added and theensuing suspension was stirred for 2 days at room temperature. Analysisby HPLC showed that two diastereomers in a ratio of 3:1 had formed.Silica gel was added to the reaction mixture and solvents wereevaporated in vacuo. The resulting solid was loaded onto a column andfurther purified by flash chromatography eluting with DCM/MeOH-NH₃ (2M)(gradient 0→5%). Combining the clean product fractions yielded 210 mg ofthe diastereomerically enriched mono-S_(N)Ar product. MS (ES) 313 (M+H),311 (M−H).

Example 11 Synthesis ofN-(2-Chloro-5-fluoropyrimidin-4-yl)-octahydro-3,3-dimethylindolizin-7(1H)-amine

Hexahydro-3,3-Dimethylindolizin-7(1H)-One

3,4-Dihydro-2,2-diemthyl-2H-pyrrole (900 mg, 9.3 mmol) was added toacetonitrile (90 ml) in a 250 ml round bottom flask, fitted with watercondenser. ZnCl₂ (0.5M) in THF (23 ml, 11.6 mmol) was added to thesolution and warmed to 60° C. 2-(Trimethylsiloxy)-1,3-butadiene (2.6 g,mmol, 18.4 mmol) was added to the mixture and heated at 90° C.overnight. The solution was cooled to room temperature and diluted withdichloromethane (90 ml) and 1N HCl (90 ml). The two layers wereseparated, the aqueous layer was basified with NH₃ (28%) in H₂O, andextracted with dichloromethane. The organic layer was washed with brineand dried with Na₂SO₄. Solid was removed by filtration and mother liquorwas concentrated in vacuo to give 250 mg brown color oil. This oil wasfurther purified by column chromatography (100% ethyl acetate to 95%ethyl acetate: 5% methanol) to give a light yellow oil (170 mg, 11%yield).

¹H NMR (300 MHz; d₆-DMSO) δ 3.05-2.99 (m, 1H), 2.69-2.63 (m, 1H),2.44-2.11 (m, 5H), 1.87-1.78 (m, 1H), 1.69-1.54 (m, 2H), 1.41-1.31 (m,1H), 1.13 (s, 3H), 0.87 (s, 3H); ¹³C NMR (300 MHz; d₆-DMSO) δ 209.7,60.44, 48.69, 42.63, 41.17, 39.67, 38.60, 28.83, 28.10, 19.87;m/z=168.08 (M+H)⁺.

Hexahydro-3,3-Dimethylindolizin-7(1H)-Ketoxime

Hexahydro-3,3-dimethylindolizin-7(1H)-one (825 mg, 4.9 mmol) was addedto a mixture of pyridine (8 ml) and ethanol (8 ml). Hydroxylamine-HClsalt (412 mg, 5.9 mmol) was added to the solution and the mixture washeated at 75° C. over weekend. The mixture was cooled to roomtemperature and concentrated under reduced pressure. N-heptane (20 ml)was added to the residue and concentrated in vacuo, this was repeatedtwice to remove most of the pyridine. Ethanol (10 ml) was added to theresidue to form a milky mixture, this was sonicated, filtered and thewhite color solid collected was dried under reduced pressure to give theproduct (613 mg, 68% yield).

¹H NMR (300 MHz; d₆-DMSO) δ 10.85 (s, 1H), 3.46-3.42 (m, 1H), 3.35-3.31(m, 1H), 2.88-2.78 (m, 2H), 2.67-2.61 (m, 1H), 2.44-2.35 (m, 2H),2.17-2.1 (m, 1H), 1.99-1.85 (m, 2H), 1.8-1.7 (m, 1H), 1.51 (s, 3H), 1.16(s, 3H); m/z=184.04 (M+H)⁺.

Octahydro-3,3-Dimethylindolizin-7(1H)-Amine

Hexahydro-3,3-diemthylindolizin-7(1H)-ketoxime (613 mg, 3.4 mmol) wasadded to acetic acid (30 ml) and followed by Pt₂O (76 mg) and was shookunder 60 psi of H₂ overnight. Addition 34 mg of Pt₂O was added to ensurethe completion of the reaction. Methanol (30 ml) was added to themixture and the catalyst was filtered off through a bed of Celite. Themother liquor was concentrated in vacuo to give a dark brown oil. Ethylacetate (50 ml) was added to the oil and it was passed through a bed ofCelite to remove the residual catalyst. The mother liquor wasconcentrated in vacuo to give 720 mg of product as an acetate salt andwas used for the next step without further purification.

¹H NMR (300 MHz; d₆-DMSO) δ 2.70-2.67 (m, 1H), 2.34-2.25 (m, 1H),2.05-1.98 (m, 1H), 1.82-1.66 (m, 2H), 1.53-1.45 (m, 3H), 1.22-1.1 (m,2H), 1.02 (s, 3H), 0.81 (s, 3H), m/z=169.09 (M+H)⁺.

¹H NMR (300 MHz; d₆-DMSO) δ 8.19 (s, 1H), 8.07 (d, J=7.57 Hz, 1H), 7.68(bd, J=5.4 Hz, 1H), 4.14 (s, 1H), 2.75-2.72 (m, 1H), 2.61-2.57 (m, 1H),2.02-1.97 (m, 1H), 1.87-1.82 (m, 1H), 1.71-1.66 (m, 2H), 1.53-1.48 (m,2H), 1.43-1.33 (m, 1H), 1.22-1.17 (m, 1H), 1.06 (s, 3H), 0.88 (s, 3H);m/z=299.09 (M+H)⁺; m/z=297.09 (M−H)⁺.

Example 12 Synthesis ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-amine)pyrimidine-2,4-diamine(Compounds 1-4)

Compound 1 is a mixture of four isomers. Compound 2 is a singleenantiomer separated by chiral HPLC. Compound 3 is a single enantiomerseparated by chiral HPLC. Compound 4 is a mixture of two enantiomers (noseparation).

The preparation of Compound 1, Compound 2, Compound 3, and Compound 4 isillustrated in accompanying scheme. In the second SNAr reaction thediastereomeric mixture (D1:D2=1:3) of(RS,SR,RR,SS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine was used and chiral HPLC was performed onthe final product. Absolute stereochemistry of the individual isomerswas not established in this series.

A solution of(R/S,S/R,R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine (0.12 g, 0.4 mmol, mixture of bothdiastereomers 1:3) in 3 ml of iPrOH,4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-amine (0.19 g, 0.5mmol; prepared according to WO2010/083207 pages 73-74, which is herebyincorporated by reference in its entirety) and 4N HCl in dioxane (0.1ml) were added. The reaction mixture was heated to 100° C. in a sealedvial for 4 hours. LCMS analysis of the crude reaction mixture indicatedthe completion of the reaction. The crude product was purified by columnchromatography to give 0.18 g (95% yield) of the product as a mixture offour stereoisomers. Two enantiomers (Compounds 2 and 3) were obtained assingle enantiomers after chiral HPLC purification. Compound 4 wasobtained as a pure diastereomer (mixture of two enantiomers). Absolutestereochemistry of individual isomers was not determined.

Compound 2: (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz) δ: 9.40 (s, 1H), 8.43 (s, 1H), 7.92 (d, J=8.9Hz, 1H), 7.55-7.7.63 (m, 2H), 7.19 (d, J=8.9 Hz, 1H), 4.38 (br. m, 1H),3.08 (m, 1H), 2.18-2.30 (m, 2H), 1.92-1.98 (m, 1H), 1.80-1.90 (m, 3H),1.74 (s, 6H), 1.38-1.70 (m, 4H), 1.20 (s, 3H), 1.32 (s, 3H); LCMS (m/z):480 (MH⁺). Chiral HPLC Pk1 RT=14.91 min. (see protocol-5 in generalmethods).

Compound 3: (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz) δ: 9.40 (s, 1H), 8.43 (s, 1H), 7.92 (d, J=8.9Hz, 1H), 7.55-7.63 (m, 2H), 7.19 (d, J=8.9 Hz, 1H), 4.38 (br. m, 1H),3.08 (m, 1H), 2.18-2.30 (m, 2H), 1.92-1.98 (m, 1H), 1.80-1.90 (m, 3H),1.74 (s, 6H), 1.38-1.70 (m, 4H), 1.20 (s, 3H), 1.32 (s, 3H); LCMS (m/z):480 (MH⁺) (see protocol-1 in general methods). Chiral HPLC Pk1 RT=17.64min. (see protocol-4 in general methods)

Compound 4: (Mixture of Two Enantiomers)

¹H NMR (DMSO d₆, 300 MHz) δ: 9.51 (s, 1H), 8.72 (s, 1H), 7.99 (d, J=8.7Hz, 1H), 7.50-7.65 (m, 2H), 7.15 (d, J=8.7 Hz, 1H), 4.35 (br. s, 1H),3.18 (m, 1H), 1.92-2.35 (m, 6H), 1.75 (s, 3H), 1.72 (s, 3H), 1.50-1.68(m, 4H), 1.37 (s, 3H), 1.28 (s, 3H); LCMS (m/z): 480 (MH⁺) (seeprotocol-1 in general methods). Chiral HPLC Pk1 RT=19.19 min. (seeprotocol-4 in general methods).

Example 13 Synthesis of(R/S,S/R,R/R,S/S)—N²-(4-(1-isopropylpiperidin-4-yloxy)-3-(difluoromethoxy)phenyl)-5-fluoro-N⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compounds 6-10)

Synthesis of Compound 6, Compound 7, Compound 8, Compound 9, andCompound 10 is illustrated in the accompanying scheme. In the 2^(nd)SNAr reaction the diastereomeric mixture (D1:D2=1:3) of(R/S,S/R,R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-aminewas used and chiral HPLC was performed on the final product. Absolutestereochemistry of the individual isomers was not established in thisseries.

A solution of(R/S,S/R,R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(0.12 g, 0.40 mmol, mixture of both diastereomers 1:3) in 3 ml of iPrOH,4-(1-isopropylpiperidin-4yloxy)-3-(difluoromethoxy)benzeneamine (0.144g, 0.48 mmol;) and 4N HCl in dioxane (0.1 ml) were added. The reactionmixture was heated to 100° C. in a sealed vial for overnight. LCMSanalysis of the crude reaction mixture indicated the completion of thereaction. The crude product was purified by column chromatography togive 0.15 g (63% yield) of the product as a mixture of four isomers(Compound 6, (two diastereomers and each diastereomer is a mixture of 2enantiomers). Chiral HPLC purification of the mixture gave all fourisomers (Compound 7, Compound 8, Compound 9 and Compound 10). Absolutestereochemistry of the individual isomers was not determined.

Compound 7: (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz): δ 9.39 (s, 1H), 8.47 (s, 1H), 8.11 (s, 1H),7.94 (d, J=2.8 Hz, 1H), 7.56-7.70 (m, 3H), 7.14 (d, J=8.8 Hz, 1H), 5.74(s, 1H), 4.32-4.40 (br. m, 1H), 4.11-4.13 (m, 1H), 3.08-3.15 (m, 4H),2.70-2.81 (m, 2H), 2.44 (s, 3H), 2.21-2.27 (m, 1H), 1.90-1.97 (m, 2H),1.69-1.75 (m, 1H), 1.51-1.55 (m, 2H), 1.32 (d, J=6.60 Hz, 6H), 1.26-1.41(m, 2H), 1.13-1.18 (m, 1H), 0.95 (s, 3H), 0.90 (s, 3H); LCMS (m/z): 563(MH⁺). Chiral HPLC Pk1 RT=16.22 min. (see protocol-5 in generalmethods).

Compound 8: (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz): δ 9.42 (s, 1H), 8.66 (s, 1H), 8.11 (s, 1H),7.92 (d, J=2.9 Hz, 1H), 7.35-7.61 (m, 3H), 7.09 (d, J=8.9 Hz, 1H), 5.68(s, 1H), 4.32-4.40 (br. s, 1H), 4.05-4.06 (m, 1H), 3.02-3.09 (m, 4H),2.65-2.78 (m, 2H), 2.35 (s, 3H), 2.19-2.25 (m, 1H), 1.88-1.37 (m, 2H),1.65-1.70 (m, 1H), 1.50-1.54 (m, 2H), 1.31 (d, J=6.6 Hz, 6H), 1.20-1.35(m, 2H), 1.10-1.16 (m, 1H), 0.93 (s, 3H), 0.87 (s, 3H); LCMS (m/z): 563(MH⁺). Chiral HPLC Pk2 RT=21.28 min. (see protocol-5 in generalmethods).

Compound 9: (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz): δ 9.45 (s, 1H), 8.40 (s, 1H), 8.25 (s, 1H),7.90 (d, J=2.7 Hz, 1H), 7.55-7.70 (m, 3H), 7.18 (d, J=8.5 Hz, 1H), 5.72(s, 1H), 4.30-4.40 (m, 1H), 4.01-4.10 (m, 1H), 3.05-3.16 (m, 4H),2.75-2.82 (m, 2H), 2.46 (s, 3H), 2.20-2.23 (m, 1H), 1.95-2.15 (m, 2H),1.69-1.75 (m, 1H), 1.50-1.65 (m, 2H), 1.42 (d, J=6.5 Hz, 6H), 1.26-1.41(m, 2H), 1.14-1.17 (m, 1H), 1.10 (s, 3H), 0.85 (s, 3H); LCMS (m/z): 563(MH⁺). Chiral HPLC Pk3 RT=27.28 min. (see protocol-5 in generalmethods).

Compound 10: (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz): δ 9.43 (s, 1H), 8.39 (s, 1H), 8.24 (s, 1H),7.85 (d, J=2.5 Hz, 1H), 7.54-7.64 (m, 3H), 7.20 (d, J=8.5 Hz, 1H), 5.71(s, 1H), 4.29-4.35 (m, 1H), 4.10-4.15 (m, 1H), 3.05-3.16 (m, 4H),2.73-2.80 (m, 2H), 2.43 (s, 3H), 2.20-2.23 (m, 1H), 1.95-2.15 (m, 2H),1.70-1.75 (m, 1H), 1.61-1.65 (m, 2H), 1.45 (d, J=6.50 Hz, 6H), 1.25-1.40(m, 2H), 1.15-1.18 (m, 1H), 1.09 (s, 3H), 0.89 (s, 3H); LCMS (m/z): 563(MH⁺). Chiral HPLC Pk 4 RT=32.53 min. (see protocol-5 in generalmethods).

Example 14 Synthesis of(R/S,S/R,R/R,S/S)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-yl)N2-(3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)phenyl)pyrimidine-2,4-diamine(Compounds 11-14)

Synthesis of Compound 11, Compound 12, Compound 13 and Compound 14 wasdescribed in the accompanying scheme. In the 2^(nd) SNAr reaction thediastereomeric mixture (D1:D2=1:3) of(R/S,S/R,R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-aminewas used and chiral HPLC was performed on the final product. Absolutestereochemistry of the individual isomers was not established.

A solution of(R/S,S/R,R/R,S/S)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(0.100 g, 0.34 mmol, mixture of two diastereomer (1:3)) in 3 ml ofiPrOH, 3-methoxy-5-(5-methyl-1H-tetrazol-1-yl)aniline (0.077 g, 0.4mmol; ChemBridge Building Blocks) and 4N HCl in dioxane (0.1 ml) wereadded. The reaction mixture was heated to 100° C. in a sealed vial for12 hours. LCMS analysis of the crude reaction mixture indicated thecompletion of the reaction. The crude product was purified by columnchromatography to give 0.14 g (Yield=88%) of the product as a mixture offour isomers (two diastereomers and each diastereomer is a mixture of 2enantiomers). Chiral HPLC purification of the mixture gave all fourisomers (Compound 11, Compound 12, Compound 13 and Compound 14).Absolute stereochemistry of the individual isomers was not established.

Compound 11: Single Diastereomer; Single Enantiomer

¹H NMR (DMSO d₆, 300 MHz) δ: 9.98 (s, 1H), 9.42 (s, 1H), 8.11 (s, 1H),7.88 (d, J=3.9 Hz, 1H), 7.33 (s, 1H), 6.98 (d, J=5.5 Hz, 1H), 6.94 (s,1H), 4.19 (br. s, 1H), 3.75 (s, 3H), 2.72-2.82 (m, 2H), 2.45 (s, 3H),2.21-2.25 (m, 1H), 1.92-1.98 (m, 2H), 1.71-1.75 (m, 1H), 1.49-1.56 (m,2H), 1.28-1.39 (m, 2H), 1.15-1.20 (m, 1H), 0.92 (s, 3H), 0.90 (s, 3H);LCMS (m/z): 468 (MH⁺); Chiral HPLC Pk1 RT=17.92 min. (see protocol-5 ingeneral methods).

Compound 12: Single Diastereomer; Single Enantiomer

¹H NMR (DMSO d₆, 300 MHz) δ: 9.98 (s, 1H), 9.42 (s, 1H), 8.11 (m, 1H),7.87 (d, J=3.9 Hz, 1H), 7.33 (m, 1H), 6.94 (m, 2H), 4.12 (br. m, 1H),3.75 (s, 3H), 2.70-2.81 (m, 2H), 2.44 (s, 3H), 2.21-2.27 (m, 1H),1.90-1.97 (m, 2H), 1.70-1.76 (m, 1H), 1.50-1.57 (m, 2H), 1.26-1.41 (m,2H), 1.13-1.18 (m, 1H), 0.91 (s, 3H), 0.88 (s, 3H); LCMS (m/z): 468(MH⁺); Chiral HPLC Pk2 RT=23.56 min. (see protocol-5 in generalmethods).

Compound 13: Single Diastereomer; Single Enantiomer

¹H NMR (DMSO d₆, 300 MHz) δ: 9.99 (s, 1H), 9.32 (s, 1H), 7.91 (s, 1H),7.83 (d, J=3.9 Hz, 1H), 7.40 (s, 1H), 7.25 (d, J=8.5 Hz, 1H), 6.91 (s,1H), 4.01-4.16 (br. m, 1H), 2.73 (m, 1H), 3.74 (s, 3H), 2.44 (s, 3H),2.16-2.20 (m, 2H), 1.91-2.03 (m, 1H), 1.49-1.70 (m, 4H), 1.30-1.40 (m,1H), 1.02-1.19 (m, 2H), 0.99 (s, 3H), 0.73 (s, 3H); LCMS (m/z): 468(MH⁺); Chiral HPLC Pk3 RT=28.18 min. (see protocol-5 in generalmethods).

Compound 14: Single Diastereomer; Single Enantiomer

¹H NMR (DMSO d₆, 300 MHz) δ: 9.99 (s, 1H), 9.33 (s, 1H), 7.90 (s, 1H),7.84 (d, J=3.9 Hz, 1H), 7.39 (s, 1H), 7.26 (d, J=8.5 Hz, 1H), 6.92 (s,1H), 4.00-4.15 (br. m, 1H), 2.74 (m, 1H), 3.74 (s, 3H), 2.43 (s, 3H),2.15-2.21 (m, 2H), 1.90-2.00 (m, 1H), 1.49-1.70 (m, 4H), 1.30-1.40 (m,1H), 1.02-1.19 (m, 2H), 0.98 (s, 3H), 0.72 (s, 3H); LCMS (m/z): 468(MH⁺); Chiral HPLC Pk4 RT=33.19 min. (see protocol-5 in generalmethods).

Compounds 11 and 12 appear to be enantiomers of one another.

Compounds 13 and 14 appear to be enantiomers of one another.

Example 15 Synthesis ofN2-{4-cyclopropyl-6-fluoro-[3-(4-methyl)-1,2,3,4-tetrazol-5-one-1-yl]}phenyl-5-fluoro-N4(7-amino-hexahydro-3,3-dimethylindolizin-5(1H)-one))2,4-pyrimidinediamine (Compound 30)

To a solution of1-(5-(4-chloro-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(described in WO 2011/068898, published Jun. 9, 2011, which is herebyincorporated by reference in its entirety; 0.11 g, 0.3 mmol) in iPrOH(10 ml), diisopropyl ethyl amine (0.21 ml, 1.2 mmol) and7-amino-hexahydro-3,3-dimethylindolizin-5(1H)-one (0.060 g, 0.329 mmol)were added. The reaction mixture was heated at 100° C. for 12 hours.LC-MS analysis indicated the completion of reaction. Volatiles wereremoved under vacuum and the crude product was adsorbed on silica gel.The crude product was purified by column chromatography to give theproduct white powder in 75% yield (0.12 g).

¹H NMR (DMSO d₆, 300 MHz) δ: 8.61 (s, 1H), 8.02 (d, J=7.6 Hz, 1H), 7.85(d, J=3.8 Hz, 1H), 7.78 (d, J=6.7 Hz, 1H), 6.97 (d, J=12.3 Hz, 1H), 4.33(br. s, 1H), 3.60-3.68 (m, 4H), 2.27 (d, J=17.0 Hz, 1H), 2.16 (d, J=12.0Hz, 1H), 1.79-1.85 (m, 1H), 1.58-1.72 (m, 3H), 1.46 (s, 3H), 1.35-1.43(m, 3H), 1.32 (s, 3H), 0.80-0.83 (m, 2H), 0.63-0.65 (m, 2H); ¹⁹F NMR(DMSO) δ: −165.08, −121.69.; LCMS (m/z): 526 (MH⁺).

Example 16 Synthesis of1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizi-7-yl-amino)-5-fluoropyrimidin-2-yl-amino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5-(4H)-one(Compound 17)

A mixture of(7R,8aS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Compound 3-10 from Example 3) (10.8 g, 36.2 mmol),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(12.6 g, 50.6 mmol) and para-toluenesulfonic acid monohydrate (12.4 g,65.1 mmol) in ^(i)PrOH (120 mL) was heated to reflux and stirred for 16hours. After allowing the reaction mixture to cool, EtOAc (600 mL) wasadded and the resulting mixture was washed with 1N NaOH (200 mL). Theorganic and aqueous layers were partitioned and the aqueous layerextracted with EtOAc (200 mL). The combined organic extracts were dried(MgSO₄), filtered and the solvent removed in vacuo to leave a cruderesidue. The residue was purified by column chromatography on silica gelusing CH₂Cl₂/2N NH₃ in MeOH (95:5) as eluent to give the product (12.7g) containing a trace of TsOH. The product was dissolved in EtOAc (500mL) and washed with 1N NaOH (100 mL) and H₂O (100 mL). The organic layerwas dried (MgSO₄) and concentrated in vacuo to give the product (12.1 g)as a solid.

[α]_(D)=−7.4 (c=0.25 in MeOH)

¹H NMR (DMSO-d₆, 300 MHz): δ 8.48 (br. s, 1H), 7.96 (d, J=7.8 Hz, 1H),7.82 (d, J=3.6 Hz, 1H), 7.25 (d, J=8.4 Hz, 1H), 6.98 (d, J=12.3 Hz, 1H),4.05-3.90 (m, 1H), 3.61 (s, 3H), 2.81 (td, J=8.4, 3.0 Hz, 1H), 2.21 (q,J=8.1 Hz, 1H), 2.20-2.10 (m, 1H), 2.00-1.90 (m, 1H), 1.75-1.48 (m, 5H),1.38 (t, J=12.3 Hz, 1H), 1.22-1.12 (m, 1H), 1.03 (s, 3H), 1.08-1.00 (m,1H), 0.83-0.77 (m, 2H), 0.75 (s, 3H), 0.63-0.57 (m, 2H)

¹⁹F NMR (DMSO-d₆, 282 MHz): δ −121.04 (t), −166.01 (s)

m/z=511.0 (M+H)⁺

The enantiomeric excess of the product is measured by chiral HPLC usingthe conditions detailed below.

Chiral HPLC Conditions:

-   -   Column: Daicel Chemical Industries, Chiralcel OJ, 4.6×250 mm    -   Mobile phase: 2:2:1 Methanol/Ethanol/Hexane 0.1% triethylamine        (isocratic)    -   Flow rate: 0.5 ml/min    -   Run time: 15 minutes    -   Temperature: room temperature    -   Detection: Water 996 PDA    -   HPLC: Waters 2690 Separations Module        Large-Scale Reaction Avoiding Use of Column Chromatography:

1-(5-Amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(26.7 g, 107.3 mmol) and para-toluene sulfonic acid monohydrate (30.6 g,161.0 mmol) were added to a stirred mixture of chloro-pyrimidine(Compound 3-10 from Example 3) (23.0 g, 68.25 mmol) in ^(i)PrOH (300mL). The reaction mixture was heated to reflux and stirred for 16 hours,after which LC-MS analysis indicated completion of the reaction. Thereaction mixture was cooled to room temperature and filtered to obtain atan-colored solid. The filter cake was then washed with ^(i)PrOH (1×25mL). The filtrate was concentrated to ca. half volume in vacuo and theemerging precipitate was filtered. The combined filter cakes from theabove procedures were air-dried then dissolved in EtOAc (300 mL) andwashed with 1N NaOH (1×200 mL then 3×100 mL). The organic layer wasdried over (MgSO₄), filtered and solvent removed in vacuo to leave theproduct (27.2 g, 78%) as a white powder.

Example 17 Synthesis ofOctahydro-5,5-dimethylindolizin-7ylamino)-5-fluoropyrimidin-2-yl-amino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compounds 16-21)

The synthesis of Compounds 16, 17, 18, 19, 20, and 21 is illustrated inthe accompanying schemes. First the separation of diastereomers D1 andD2 was accomplished by column chromatography and both the diastereomerswere taken to the SNAr reaction separately. The final products wereseparated by chiral HPLC provide individual enantiomers.

(R/S,S/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(D1) is a racemic mixture of two enantiomers. D1 was taken to 2^(nd)SNAr reaction to form Compound 16. Synthesis of Compound 16 (racemic)was described in the accompanying scheme. Compound 16 was separated bychiral HPLC to give Compound 17 (Single enantiomer) and Compound 18(Single Enantiomer).

To a solution of(R/S,S/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(D1) (0.1 g, 0.3 mmol) in 10 ml of iPrOH,1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(0.092 g, 0.37 mmol) and p-toluene sulfonicacid monohydrate (0.064 g,0.3 mmol) were added. The reaction mixture was heated at 120° C. (bathtemp.) for 16 hours. LC-MS analysis indicated the completion of thereaction. The reaction mixture was cooled to room temperature andfiltered off to get tan solid. The filter cake was washed with 5 ml ofiPrOH to get white solid. The filtrate was concentrated in vacuo to halfthe volume and the obtained solids were filtered off. The filteredsolids were combined and dried under air. The dried solids weredissolved in 50 ml of EtOAc and partitioned with aqueous 1N NaOH (10ml). Both the layers were separated and organic layer was washed (×3)with aqueous 1N NaOH (10 ml). Separated organic layer was dried overMgSO₄ and concentrated to give 0.12 g (yield=71%) of the product aswhite powder (Compound 16). Compound 16 was separated by chiral HPLC togive the corresponding enantiomers Compound 17 and Compound 18(seeProtocol-3 in general methods).

1-(5-(4-((7R,8aS)-Octahydro-5,5-dimethylindolizin-7ylamino)-5-fluoropyrimidin-2-yl-amino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 17) (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz) δ: 8.48 (s, 1H), 7.97 (d, J=7.6 Hz, 1H), 7.82(d, J=3.8 Hz, 1H), 7.25 (d, J=7.9 Hz, 1H), 6.99 (d, J=12.0 Hz, 1H),3.90-4.10 (br. m, 1H), 3.61 (s, 3H), 2.81 (dt, J=8.2, 2.9 Hz, 1H),2.10-2.26 (m, 2H), 1.93 (d, J=11.7 Hz, 1H), 1.49-1.75 (m, 5H), 1.39 (t,J=12.3 Hz, 1H), 1.14-1.24 (m, 1H), 1.03 (s, 3H), 0.94-1.01 (m, 1H),0.77-0.83 (m, 2H), 0.75 (s, 3H), 0.59-0.63 (m, 2H); ¹⁹F NMR (DMSO) δ:−165.99 (d), −121.09 (t).; LCMS (m/z): 512 (MH⁺); Chiral HPLC RT=18.89min. (see Protocol-4 in general methods).

1-(5-(4-((7S,8aR)-Octahydro-5,5-dimethylindolizin-7ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 18) (Single Enantiomer)

¹H NMR (DMSO d₆, 300 MHz) δ: 8.48 (s, 1H), 7.97 (d, J=7.6 Hz, 1H), 7.83(d, J=3.8 Hz, 1H), 7.25 (d, J=8.2 Hz, 1H), 6.99 (d, J=12.3 Hz, 1H),3.90-4.05 (br. m, 1H), 3.61 (s, 3H), 2.81 (br. m, 1H), 2.10-2.26 (m,2H), 1.93 (d, J=11.7 Hz, 1H), 1.49-1.75 (m, 5H), 1.39 (t, J=12.0 Hz,1H), 1.14-1.24 (m, 1H), 1.03 (s, 3H), 0.94-1.01 (m, 1H), 0.77-0.82 (m,2H), 0.75 (s, 3H), 0.58-0.63 (m, 2H); ¹⁹F NMR (DMSO) δ: −166.00 (d),−121.05 (t); LCMS (m/z): 512 (MH⁺). Chiral HPLC RT=22.97 (see Protocol-4in general methods).

(S/S,R/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(D2) is a mixture of two enantiomers. D2 was taken to 2^(nd) SNArreaction to form Compound 19. Synthesis of Compound 19 (mixture of twoenantiomers) was described in the accompanying scheme. Compound 19 wasseparated by chiral HPLC to give Compound 20 (Single Enantiomer) andCompound 21 (Single Enantiomer).

To a solution of (S/S,R/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine (D2) (0.10 g, 0.34 mmol) in 10 ml of iPrOH,1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(0.092 g, 0.4 mmol) and p-toluene sulfonic acid monohydrate (0.064 g,0.3 mmol) were added. The reaction mixture was heated at 120° C. (bathtemp.) for 16 hours. LC-MS analysis indicated the completion of thereaction. The reaction mixture was cooled to room temperature andfiltered off to get tan solid. The filter cake was washed with 1 ml ofiPrOH to get white solid. The filtered solids were combined and driedunder air. The dried solids were dissolved in 50 ml of EtOAc andpartitioned with aqueous 1N NaOH (10 ml). Both the layers were separatedand organic layer was washed (×3) with aqueous 1N NaOH (10 ml).Separated organic layer was dried over MgSO₄ and concentrated to give0.13 g (yield=74%) of the product as white powder (Compound 19).Compound 19 was separated by chiral HPLC to give the correspondingenantiomers Compound 20 and Compound 21 (see Protocol-4 in generalmethods).

Compound 20: (Single Enantiomer):

¹H NMR (DMSO d₆, 300 MHz) δ: 8.56 (s, 1H), 8.11 (d, J=7.6 Hz, 1H), 7.84(d, J=3.8 Hz, 1H), 6.92-6.99 (m, 2H), 4.04 (br. s, 1H), 3.61 (s, 3H),2.70-2.83 (m, 2H), 2.25-2.35 (m, 1H), 1.75-1.95 (m, 3H), 1.55-1.65 (m,3H), 1.15-1.36 (m, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.78-0.83 (m, 2H),0.60-0.65 (m, 2H); ¹⁹F NMR (DMSO) δ: −165.78 (d), −122.24 (t); LCMS(m/z): 512 (MH⁺); Chiral HPLC RT=29.88 min. (see Protocol-4 in generalmethods).

Compound 21: (Single Enantiomer):

¹H NMR (DMSO d₆, 300 MHz) δ: 8.57 (s, 1H), 8.11 (d, J=7.6 Hz, 1H), 7.84(d, J=3.8 Hz, 1H), 6.92-6.99 (m, 2H), 4.04 (br. s, 1H), 3.61 (s, 3H),2.71-2.83 (m, 2H), 2.25-2.35 (m, 1H), 1.76-1.95 (m, 3H), 1.56-1.68 (m,3H), 1.15-1.35 (m, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.78-0.83 (m, 2H),0.60-0.65 (m, 2H); ¹⁹F NMR (DMSO) δ: −165.57 (d), −122.24 (t); LCMS(m/z): 512 (MH⁺); Chiral HPLC RT=34.50 min. (see Protocol-4 in generalmethods).

Example 18 Synthesis ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compound 5)

Preparation of8-Fluoro-2,2-dimethyl-6-nitro-2H-benzo[b][1,4]oxazin-3(4H)-thione

To a solution of8-Fluoro-2,2-dimethyl-6-nitro-2H-benzo[b][1,4]oxazin-3(4H)-one (0.75 g,3.1 mmol) in 25 ml anhydrous THF, P₂S₅ (2.8 g, 6.25 mmol) was added andthe reaction mixture was heated to reflux for 12 hours. Analysis ofLC-MS indicated the completion of the reaction. Volatiles were removedin vacuo and the crude reaction mixture was partitioned in EtOAc (100ml) and saturated aqueous NH₄Cl (100 ml). Layers were separated and theorganic layer was washed (×2) with saturated aqueous NH₄Cl (50 ml).EtOAc layer was dried over Na₂SO₄ and concentrated to give the productin 69% yield (0.55 g). LCMS (m/z) 257 (MH⁺).

Preparation of4,4-dimethyl-6-fluoro-8-nitro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazine

To a solution of8-fluoro-2,2-dimethyl-6-nitro-2H-benzo[b][1,4]oxazin-3(4H)-thione (1.0g, 3.9 mmol) in THF, Hg(OAc)₂ was added at 0° C. TMS-N₃ was added to thereaction mixture at 0° C. and stirred for 1 hour. Reaction mixture wasallowed to warm to room temperature and stirred for 3 hours. LCMSanalysis of the reaction mixture indicated completion of the reaction.After 3 hours, the reaction mixture was diluted with 100 ml of EtOAc andpartitioned with saturated aqueous NH₄Cl (100 ml). Layers were separatedand the aqueous layer was washed twice with EtOAc (50 ml). CombinedEtOAc layers were dried over Na₂SO₄ and concentrated. The crude productwas purified by column chromatography to give the product in 57% yield(0.57 g). LCMS (m/z) 266 (MH⁺).

Preparation of4,4-dimethyl-6-fluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-amine

4,4-Dimethyl-6-fluoro-8-nitro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazine(0.82 g, 3.1 mmol) is dissolved in EtOH (20 ml). The clear solution istransferred to a Parr hydrogenation flask and placed under nitrogen. 10%Pd-C(0.25 g) was added to the Parr flask under nitrogen. The mixture wasthen transferred to a Parr hydrogenation apparatus, evacuated and filledwith hydrogen (×3). The mixture was hydrogenated at 30 psi (optionallytopping-up hydrogen) until LC/MS and TLC indicated complete reaction tothe amine. After complete reaction, the mixture was placed undernitrogen and filtered through a small pad of Celite. The filter cake waswashed with EtOH (×1) and the filtrate was concentrated in vacuo toleave the4,4-dimethyl-6-fluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-amine in72% yield (0.53 g). LCMS (m/z): 236 (MH⁺).

Preparation ofN2-(4,4-dimethyl-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-yl)-5-fluoro-N4-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compound 5)

A solution of(R/S,S/R)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(D1) (0.1 g, 0.3 mmol, single diastereomer) in 3 ml of iPrOH,4,4-dimethyl-6-fluoro-4H-benzo[b]tetrazolo[1,5-d][1,4]oxazin-8-amine(0.093 g, 0.4 mmol) and 4N HCl in dioxane (0.1 ml) were added. Thereaction mixture was heated to 100° C. in a sealed vial for 12 hours.LCMS analysis of the crude reaction mixture indicated the completion ofthe reaction. The crude product was purified by column chromatography togive 0.12 g (Yield=72%) of the product as a mixture of two enantiomers.

¹H NMR (DMSO d₆, 300 MHz) δ: 9.58 (s, 1H), 8.40 (s, 1H), 7.90 (d, J=8.5Hz, 1H), 7.75 (d, J=8.8 Hz, 1H), 7.10 (d, J=5.5 Hz, 1H), 4.25 (br. s,1H), 2.85 (m, 2H), 2.23-2.335 (m, 1H), 2.15 (m, 2H), 1.88 (s, 6H),1.40-1.60 (m, 4H), 1.22 (m, 2H), 1.05 (s, 3H), 0.99 (s, 3H); LCMS (m/z):498 (MH⁺).

Example 19 Synthesis of4-((R/S,S/R)-Octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-cyclopropyl-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-yl)phenylamino)pyrimidine-5-carbonitrile (Compound 66)

To a solution of(±)-2-chloro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyridimine-5-carbonitrile(70% pure, 0.068 g, 0.2 mmol) in iPrOH (10 ml),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(0.078 g, 0.3 mmol) and p-TSA (0.04 g, 0.2 mmol) were added and thereaction mixture was heated to 82° C. for overnight. LCMS analysisindicated the completion of reaction. Purification of the crude productby column chromatography gave the product as p-toluene sulfonic acidsalt. The salt was dissolved in 20 ml EtOAc and partitioned with aqueous2N NaOH solution. The organic layers were separated and dried overNa₂SO₄. Removal of the solvents and lyophilization gave the product in11% yield (0.015 g).

¹H NMR (DMSO d₆, 300 MHz) δ: 8.43 (s, 1H), 8.29 (s, 1H), 7.65 (d, J=7.3Hz, 1H), 7.42 (d, J=7.6 Hz, 1H), 7.03 (d, J=11.7 Hz, 1H), 3.99 (br. s,1H), 3.61 (s, 3H), 2.76-2.80 (m, 1H), 2.10-2.26 (m, 2H), 1.79-1.83 (m,1H), 1.52-1.70 (m, 4H), 1.34-1.43 (m, 2H), 1.07-1.22 (m, 4H), 1.01 (s,3H), 0.82-0.86 (m, 2H), 0.63-0.67 (m, 2H); LCMS (m/z): 519 (MH⁺).

Example 20 Synthesis of(±)-N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydroindolizin-7-yl)pyrimidine-2,4-diamine(Compound 24)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amine (90 mg,0.34 mmol), 4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)benzeneamine (87mg, 0.35 mmol; prepared according to U.S. patent application Ser. No.13/188,222, filed Jul. 21, 2011, now U.S. patent application publicationUS2012/0022092, which is hereby incorporated by reference in itsentirety) and para-toluenesulfonic acid monohydrate (63 mg, 0.34 mmol)in isopropyl alcohol (3 ml) were combined in a sealed vial, heated to70° C. and stirred over a couple of days. After cooling, a precipitateemerged which was filtered to give a crude solid (50 mg). The crudesolid was purified further by preparative high-performance liquidchromatography to give a solid. The solid was partitioned between EtOAc(20 ml) and 1N NaOH (20 ml). The aqueous layer was extracted with EtOAc(1×20 ml) and the combined organic extracts were dried (MgSO₄), filteredand the solvent removed under vacuum to leave the product (10 mg, 7%) asa solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.83 (t, J=0.9 Hz, 1H), 8.59 (s, 1H), 8.20(d, J=7.7 Hz, 1H), 7.80 (d, J=6.5 Hz, 1H), 7.36 (d, J=8.1 Hz, 1H), 7.04(d, J=12.2 Hz, 1H), 3.73-3.59 (m, 1H), 2.82-2.75 (m, 2H), 1.87 (q, J=8.4Hz, 1H), 1.82-1.78 (m, 2H), 1.64-1.41 (m, 5H), 1.39-1.28 (m, 2H),1.20-1.03 (m, 2H), 0.71-0.65 (m, 2H), 0.57-0.52 (m, 2H); ¹⁹F NMR (282MHz; d₆-DMSO) δ −122.1 (t), −165.5 (d); m/z=454.29 (M+H)⁺; rt=3.37 min(HPLC protocol-1).

Example 21 Synthesis of(±)-1-(5-(5-fluoro-4-(octahydroindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)one(Compound 27)

A mixture of ofN-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amine (54 mg,0.2 mmol),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5-one (52mg, 0.21 mmol; prepared according to US20110130415 pages 43-48, which ishereby incorporated by reference in its entirety) andpara-toluenesulfonic acid monohydrate (38 mg, 0.2 mmol) in isopropylalcohol (3 ml) were combined in a sealed vial, heated to 70° C. andstirred for 2 days. After cooling, the mixture was concentrated undervacuum to leave a crude residue. The residue was purified by preparativehigh-performance liquid chromatography to give a solid. The solid waspartitioned between EtOAc (20 ml) and 1N NaOH (20 ml). The aqueous layerwas extracted with EtOAc (2×20 ml) and the combined organic extractswere dried (MgSO₄), filtered and the solvent removed in vacuo to leavethe product (46 mg, 47%) as a solid after freeze-drying from MeCN/H₂O.

¹H NMR (300 MHz; d₆-DMSO) δ 8.52 (br. s, 1H), 8.08 (d, J=7.7 Hz, 1H),7.83 (dd, J=3.8, 1.2 Hz, 1H), 7.38 (br. d, J=7.7 Hz, 1H), 6.97 (d,J=12.3 Hz, 1H), 3.85-3.71 (m, 1H), 3.62 (app. d, 3H), 2.97-2.87 (m, 2H),1.93-1.89 (m, 2H), 1.75-1.50 (m, 7H), 1.31-1.15 (m, 3H), 0.83-0.77 (m,2H), 0.64-0.59 (m, 2H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −122.0 (t), −165.8(d); m/z=484.27 (M+H)⁺; rt=3.22 min (HPLC protocol-1).

Example 22 Synthesis of(±)-N²-(4-cyclopropyl-2-fluoro-5-(5-methyl-1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydroindolizin-7-yl)pyrimidine-2,4-diamine(Compound 28)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)octahydroindolizin-7-amine (179 mg,0.66 mmol),1-(2-Cyclopropyl-4-fluoro-5-nitrophenyl)-5-methyl-1H-tetrazole (140 mg,0.66 mmol; prepared according to U.S. patent application Ser. No.13/188,222, now U.S. patent application publication US2012/0022092) andpara-toluenesulfonic acid monohydrate (126 mg, 0.66 mol) in isopropylalcohol (5 ml) were combined in a sealed vial, heated to 70° C. andstirred for 3 days. After cooling, the mixture was concentrated in vacuoand the crude residue partitioned between 1N NaOH (25 ml) and EtOAc (30ml). The aqueous layer was extracted with EtOAc (1×30 ml) and thecombined organic layers were dried (MgSO₄), filtered and the solventremoved under vacuum to leave a crude solid. The solid was purified bycolumn chromatography on silica gel (ISCO System) using 2M NH₃ inMeOH/CH₂Cl₂ (gradient system from 0:1 to 1:9) as eluent to give theproduct (190 mg, 61%) as a solid after freeze-drying from MeCN/H₂O.

¹H NMR (300 MHz; d₆-DMSO) δ 8.61 (br. s, 1H), 8.15 (d, J=7.6 Hz, 1H),7.86-7.84 (m, 1H), 7.41 (br. d, J=8.1 Hz, 1H), 7.04 (d, J=12.3 Hz, 1H),3.75-3.64 (m, 1H), 2.92-2.82 (m, 2H), 2.41 (s, 3H), 2.05-2.91 (m, 1H),1.88-1.85 (m, 2H), 1.71-1.40 (m, 6H), 1.27-1.08 (m, 3H), 0.79-0.70 (m,2H), 0.68-0.62 (m, 2H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −122.4 (t), −165.6(d); m/z=468.28 (M+H)⁺; rt=3.40 min (HPLC protocol-1).

Example 23 Synthesis of(±)-N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compound 53)

A mixture of ofN-(2-chloro-5-fluoropyrimidin-4-yl)octahydro-5,5-dimethylindolizin-7-aminehydrochloride (168 mg, 0.5 mmol),4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)benzeneamine (153 mg, 0.7mmol; prepared according to U.S. patent application Ser. No. 13/188,222,filed Jul. 21, 2011, now U.S. patent application publicationUS2012/0022092) and para-toluenesulfonic acid monohydrate (95 mg, 0.5mmol) in isopropyl alcohol (3 ml) were combined in a sealed vial, heatedto 70° C. and stirred for 4 days. After cooling, the mixture wasfiltered and the filter cake washed with cold isopropyl alcohol. Thefilter cake was suspended in EtOAc (50 ml) and 1N NaOH (50 ml) wasadded. The aqueous and organic layers were partitioned and the aqueouslayer was extracted with EtOAc (30 ml). The combined organic extractswere dried (Na₂SO₄), filtered and the solvent removed under vacuum toleave a residue, which was freeze-dried from MeCN/H₂O to give theproduct (162 mg, 67%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.86 (m, 1H), 8.59 (s, 1H), 8.09 (d, J=7.6Hz, 1H), 7.83 (d, J=3.8 Hz, 1H), 7.26 (br. d, J=8.0 Hz, 1H), 7.09 (d,J=12.1 Hz, 1H), 4.00-3.88 (m, 1H), 2.81-2.74 (m, 1H), 2.16 (q, J=8.2 Hz,1H), 1.99-1.83 (m, 2H), 1.72-1.32 (m, 6H), 1.20-0.94 (m, 2H), 1.01 (s,3H), 0.75-0.71 (m, 2H), 0.69 (s, 3H), 0.63-0.55 (m, 2H); ¹⁹F NMR (282MHz; d₆-DMSO) δ −165.7 (s), −120.9 (t); m/z=482.33 (M+H)⁺; rt=3.44 min(HPLC protocol-1).

Example 24 Resolution of(±)-N²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-n⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compounds 54 and 55)

A small quantity of racemicN²-(4-cyclopropyl-2-fluoro-5-(1H-tetraol-1-yl)phenyl)-5-fluoro-N⁴-(octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(40 mg) was purified by chiral high-performance liquid chromatographyusing the conditions below to give individual enantiomers.

Chiral HPLC Conditions:

-   -   Column: Daicel Chemical Industries, Chiracel OJ 4.6×250 mm    -   Mobile phase: 1:1 MeOH/EtOH containing 0.1% diethylamine    -   Flow rate: 0.5 ml/min    -   Run time: 30 minutes    -   Temperature: room temperature    -   Detection: Waters 996 PDA    -   HPLC: Waters 2690 Separations Module        Compound 54

Retention time of first eluting isomer=9.30 min (15 mg); Data for firsteluting enantiomer from chiral column: ¹H NMR (300 MHz; d₆-DMSO) δ 9.87(s, 1H), 8.59 (br. s, 1H), 8.08 (d, J=7.5 Hz, 1H), 7.83 (d, J=3.7 Hz,1H), 7.33-7.24 (br. s, 1H), 7.09 (d, J=12.2 Hz, 1H), 4.03-3.87 (m, 1H),2.86-2.72 (m, 1H), 2.29-2.14 (m, 1H), 2.00-1.83 (m, 2H), 1.73-1.36 (m,6H), 1.22-1.03 (m, 5H), 0.78-0.66 (m, 5H), 0.64-0.56 (m, 2H); ¹⁹F NMR(282 MHz; d₆-DMSO) δ −165.7, −120.8; m/z=482.44 (M+H)⁺; rt=3.58 min(HPLC protocol-1).

Compound 55

Retention time for second eluting isomer=11.74 min (18 mg); Data same asfor 1st eluting isomer.

Example 25 Synthesis of(±)-2-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenylamino)-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carbonitrile(Compound 56)

A mixture of(±)-2-chloro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-5-carbonitrile(60 mg of 70% pure material from previous step),4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)benzeneamine (65 mg, 0.3mmol; prepared according to U.S. patent application Ser. No. 13/188,222,filed Jul. 21, 2011, now U.S. patent application publicationUS2012/0022092) and para-toluenesulfonic acid monohydrate (37 mg, 0.2mmol) in isopropyl alcohol (3 ml) were combined in a sealed vial, heatedto 80° C. and stirred for 4 hours. After cooling, a precipitate emergedwhich was filtered. The filter cake was suspended in EtOAc (30 ml) and1N NaOH (50 ml). The aqueous and organic layers were partitioned and theaqueous layer was extracted with EtOAc (2×25 ml). The combined organicextracts were dried (MgSO₄), filtered and the solvent removed undervacuum to leave a solid. The solid was freeze-dried from MeCN/H₂O togive the product (26 mg) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.85 (m, 1H), 9.51 (br. s, 1H), 8.30 (s,1H), 7.80 (d, J=7.3 Hz, 1H), 7.44 (d, J=7.3 Hz, 1H), 7.14 (d, J=11.8 Hz,1H), 4.08-3.89 (m, 1H), 2.83-2.71 (m, 1H), 2.28-2.13 (m, 1H), 2.06-1.88(m, 1H), 1.84-1.73 (m, 1H), 1.71-1.50 (m, 3H), 1.47-1.34 (m, 2H),1.21-1.07 (m, 3H), 1.00 (s, 3H), 0.75-0.59 (m, 7H); ¹⁹F NMR (282 MHz;d₆-DMSO) δ −117.3 (t); m/z=489.34 (M+H)⁺; rt=4.01 min (HPLC protocol-1).

Example 26 Synthesis ofN2-(4-cyclopropyl-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-yl)phenylamino)-5-fluoro-N4-(Octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidine-2,4-diamine (Compound 26)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-3,3-dimethylindolizin-7-amine(Diastereomer D1 97% dr; 52 mg, 0.174 mmol, 1 equiv),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Described in: WO 2011/068898, which is hereby incorporated by referencein its entirety. 43 mg, 0.174 mmol, 1 equiv), and PTSA monohydrate (33mg, 0.174 mmol, 1 equiv) in IPA (1 ml) were heated to 100° C. for 4days. After cooling to ambient temperature, the crude mixture wasconcentrated to dryness and taken in water, EtOAc, and 1N NaOH. Thelayers were separated. The organic layer was washed with 1N NaOH (×2),dried over Na₂SO₄, filtered, and concentrated to dryness. The crudeproduct was purified by flash chromatography and eluted with DCM:2MNH₃/MeOH=100:0 to 96:4 using 1% 2M NH₃/MeOH increments to give compound1-(5-(5-fluoro-4-(octahydro-3,3-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 26) (>90% dr, 53 mg, 60%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 8.59 (s, 1H), 8.10-8.09 (d, J=8.4 Hz),7.85-7.86 (d, J=3.6 Hz), 6.95-7.00 (m, 2H), 4.11 (bs, 1H), 3.62 (s, 1H),2.42-2.71 (m, 2H), 1.49-2.00 (m, 8H), 1.01-1.22 (m, 2H), 1.03-1.06 (m,3H), 0.823-0.871 (m, 5H), 0.630-0.645 (m, 2H); ¹⁹F NMR (282 MHz;d₆-DMSO) δ −122.0 (t), −164.7 (s); m/z=512 (M+H)⁺.

Example 27 Chiral HPLC resolution of1-(5-(5-fluoro-4-(octahydro-3,3-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compounds 34 and 35)

Chiral HPLC resolution of1-(5-(5-fluoro-4-(octahydro-3,3-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 26) (>90% dr, 35 mg) to enantiomers was performed.

Chiral HPLC method: Column: Chiralcel-OJ, 4.6×250 mm, with guard. Mobilphase: 95% Hexane, 2.4% methanol, 2.5% ethanol 0.1% triethylamine. Flowrate: 0.5 ml/min. Injection volume: 3 μL. Concentration: approx 5 mg/ml.Detection: UV at 254 nm.

Compound 34

Compound 34: (>99% ee, 9.5 mg) has Rt=21.86 min.

¹H NMR (300 MHz; d₆-DMSO) δ 8.59 (s, 1H), 8.10-8.09 (d, J=8.4 Hz),7.85-7.86 (d, J=3.6 Hz), 6.95-7.00 (m, 2H), 4.11 (bs, 1H), 3.62 (s, 1H),2.42-2.71 (m, 2H), 1.49-2.00 (m, 8H), 1.01-1.22 (m, 2H), 1.03-1.06 (m,3H), 0.823-0.871 (m, 5H), 0.630-0.645 (m, 2H); ¹⁹F NMR (282 MHz;d₆-DMSO) δ −122.0 (t), −164.7 (s); m/z=512 (M+H)⁺.

Compound 35

Compound 35: (>97% ee, 10.7 mg) has Rt=26.56 min.

¹H NMR (300 MHz; d₆-DMSO) δ 8.59 (s, 1H), 8.10-8.09 (d, 1H, J=8.4 Hz),7.85-7.86 (d, 1H, J=3.6 Hz), 6.95-7.00 (m, 2H), 4.11 (bs, 1H), 3.62 (s,1H), 2.42-2.71 (m, 2H), 1.49-2.00 (m, 8H), 1.01-1.22 (m, 2H), 1.03-1.06(m, 3H), 0.823-0.871 (m, 5H), 0.630-0.645 (m, 2H);

¹⁹F NMR (282 MHz; d₆-DMSO) δ −122.0 (t), −164.7 (s); m/z=512 (M+H)⁺.

Example 28 Synthesis of1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compounds 31-33)

Preparation of1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 31)

A mixture of7-(2-chloro-5-fluoropyrimidin-4-ylamino)-hexahydro-5,5-dimethylindolizin-3(5H)-oneisomer A (cis) (50 mg),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(50 mg) and TFA (5 drops) in isopropanol (1 ml) was heated at 100° C.overnight in a sealed vial. After allowing to cool to room temperature,the solvent was removed in vacuo and the residue was purified byCombiflash chromatography (2.0 M ammonia methanol indichloromethane=0-30%) to give product as racemic mixture.

¹H NMR (300 MHz; d₆-DMSO) δ 8.55 (s, 1H), 7.88 (d, J=7.8 Hz, 1H), 7.85(d, J=3.9 Hz, 1H), 7.34 (d, J=7.8 Hz, 1H), 7.00 (d, J=12.0 Hz, 1H),4.06-4.04 (m, 1H), 3.59 (s, 3H), 2.14-1.94 (m, 5H), 1.64-1.59 (m, 1H),1.56 (s, 3H), 1.48-1.12 (m, 4H), 1.02 (s, 3H), 0.81 (dd, J=2.1, 8.7 Hz,2H), 0.61 (q, J=4.5 Hz, 2H); LC-MS: purity: 97.99%; MS (m/e): 526.39(M+H)⁺; m/z=524.33 (M−H)⁺.

Preparation of1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 32)

A mixture of7-(2-chloro-5-fluoropyrimidin-4-ylamino)-hexahydro-5,5-dimethylindolizin-3(5H)-oneisomer B (trans) (100 mg),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(100 mg) and TFA (5 drops) in isopropanol (1 ml) was heated at 100° C.overnight in a sealed vial. After allowing to cool to room temperature,the solvent was removed in vacuo and the residue was purified byCombiflash chromatography (2.0 M ammonia methanol indichloromethane=0-30%) to give product as racemic mixture.

¹H NMR (300 MHz; d₆-DMSO) δ 8.64 (s, 1H), 8.03 (d, J=7.8 Hz, 1H), 7.88(d, J=3.9 Hz, 1H), 7.57 (d, J=5.7 Hz, 1H), 6.99 (d, J=12.3 Hz, 1H), 4.22(m, 1H), 3.89 (m, 1H), 3.59 (s, 3H), 2.26-1.53 (m, 9H), 1.32 (s, 3H),1.24 (s, 3H), 0.81 (d, J=8.4 Hz, 2H), 0.62 (d, J=3.3 Hz, 2H); LC-MS:purity: 98.22%; MS (m/e): 526.37 (M+H)⁺; m/z=524.41 (M−H)⁺.

Preparation of1-(5-(5-fluoro-4-(hexahydro-5,5-dimethylindolizin-3(5H)-one-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-oneformate (Compound 33)

¹H NMR (300 MHz; d₆-DMSO) δ 8.58 (s, 1H), 8.04 (d, J=7.8 Hz, 1H), 7.86(d, J=3.9 Hz, 1H), 7.50 (d, J=7.2 Hz, 1H), 6.99 (d, J=12.3 Hz, 1H), 4.21(m, 1H), 3.90 (m, 1H), 3.59 (s, 3H), 2.26-1.54 (m, 9H), 1.32 (s, 3H),1.25 (s, 3H), 0.81 (d, J=8.1 Hz, 2H), 0.61 (d, J=3.6 Hz, 2H); LC-MS:purity: 98.65%; MS (m/e): 526.37 (M+H)⁺; m/z=524.37 (M−H)⁺.

Example 29 Preparation of Anilines for Synthesis of Compounds

Certain anilines for the synthesis of the present compounds wereprepared as illustrated in the scheme below:

Preparation of1-(2-((R)-Tetrahydrofuran-3-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one

(R)-(−)-3-Hydroxytetrahydrofuran (440 mg, 5 mmol, 2 equiv) in THF (15ml) under argon gas, was cooled to 0° C. Potassium tert-butoxide (617mg, 5.5 mmol, 2.2 equiv) was added in one portion, and the reactionmixture was stirred for 20 minutes at 0° C. Then1-(2-fluoro-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one (Described in:WO 2011/068898. 598 mg, 2.5 mmol, 1 equiv) was added in one portion, andthe reaction mixture was stirred for 10 minutes at 0° C. and allowed towarm up to ambient temperature over 2 hours. The reaction mixture wasconcentrated, and the residue was taken in DCM and water. The layerswere separated, and the organic layer was washed with water (×2), driedover Na₂SO₄, filtered, and concentrated to provide1-(2-((R)-tetrahydrofuran-3-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(723 mg, 94%) as a yellow solid that was used without furtherpurification.

¹H NMR (300 MHz; d₆-DMSO) δ 8.41-8.47 (m, 2H), 7.51-7.54 (d, 1H, J=9Hz), 5.31-5.4 (t, 1H, J=3.6 Hz), 3.88-3.93 (dd, 1H, J=4.2 Hz, J=10.5Hz), 3.70-3.74 (m, 3H), 3.62 (s, 3H), 2.20-2.27 (m, 1H), 1.89-1.93 (m,1H); m/z=308 (M+H)⁺.

Preparation of1-(2-((S)-Tetrahydrofuran-3-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one

(S)-(+)-3-Hydroxytetrahydrofuran (440 mg, 5 mmol, 2 equiv) in THF (15ml) under argon gas, was cooled to 0° C. Potassium tert-butoxide (617mg, 5.5 mmol, 2.2 equiv) was added in one portion, and the reactionmixture was stirred for 20 minutes at 0° C. Then, fluoro tetrazole (598mg, 2.5 mmol, 1 equiv) was added in one portion, and the reactionmixture was stirred for 10 minutes at 0° C. and allowed to warm up toambient temperature over 2 hours. The reaction mixture was concentrated,and the residue was taken in DCM and water. The layers were separated,and the organic layer was washed with water (×2), dried over Na₂SO₄,filtered, and concentrated to provide1-(2-((S)-tetrahydrofuran-3-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(764 mg, 98%) as a yellow solid that was used without furtherpurification.

¹H NMR (300 MHz; d₆-DMSO) δ 8.41-8.47 (m, 2H), 7.51-7.54 (d, 1H, J=9Hz), 5.31-5.4 (t, 1H, J=3.6 Hz), 3.88-3.93 (dd, 1H, J=4.2 Hz, J=10.5Hz), 3.70-3.74 (m, 3H), 3.62 (s, 3H), 2.20-2.27 (m, 1H), 1.89-1.93 (m,1H); m/z=308 (M+H)⁺.

Preparation of1-(2-((R)-Tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one

A round-bottom flask was charged with1-(2-((R)-tetrahydrofuran-3-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(723 mg, 2.35 mmol), EtOH (25 ml), and 10% Pd/C (50% in water, Degussatype E101; 145 mg, 20 wt % by weight of the starting nitro compound)giving a suspension. The flask was sealed with a rubber septum,degassed, and back-filled with H₂ (×3) from a balloon filled with H₂.The reaction mixture was stirred for 2 hours using a H₂ filled balloon.The reaction mixture was filtered through a pad of Celite, and the padof Celite was rinsed with MeOH. The filtrate was evaporated to drynessto provide1-(2-((R)-tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one(646 mg, 99%) as a dark-brown solid that was used without furtherpurification.

¹H NMR (300 MHz; d₆-DMSO) δ 6.96-6.99 (m, 1H), 6.89-6.73 (m, 1H),6.55-6.60 (m, 1H), 5.06 (bs, 2H), 4.77-4.85 (m, 1H), 3.55-3.77 (m, 7H),1.94-2.03 (m, 1H), 1.78-1.86 (m, 1H); m/z=278 (M+H)⁺.

Preparation of1-(2-((S)-Tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one

A round-bottom flask was charged with1-(2-((S)-tetrahydrofuran-3-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(764 mg, 2.49 mmol), EtOH (25 ml), and 10% Pd/C (50% in water, Degussatype E101; 153 mg, 20 wt % by weight of the starting nitro compound)giving a suspension. The flask was sealed with a rubber septum,degassed, and back-filled with H₂ (×3) from a balloon filled with H₂.The reaction mixture was stirred for 2 hours using a H₂ filled balloon.The reaction mixture was filtered through a pad of Celite, and the padof Celite was rinsed with MeOH. The filtrate was evaporated to drynessto provide1-(2-((5)-tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one(677 mg, 98%) as a dark-brown solid that was used without furtherpurification.

¹H NMR (300 MHz; d₆-DMSO) δ 6.96-6.99 (m, 1H), 6.89-6.73 (m, 1H),6.55-6.60 (m, 1H), 5.06 (bs, 2H), 4.77-4.85 (m, 1H), 3.55-3.77 (m, 7H),1.94-2.03 (m, 1H), 1.78-1.86 (m, 1H); m/z=278 (M+H)⁺.

Preparation of 5-Nitro-2-(oxetan-3-yloxy)benzonitrile

General procedure: 3-Hydroxy oxetane (370 mg, 5 mmol, 2 equiv) in THF(15 ml) under argon gas, was cooled to 0° C. Potassium tert-butoxide(617 mg, 5.5 mmol, 2.2 equiv) was added in one portion, and the reactionmixture was stirred for 20 minutes at 0° C. Then2-fluoro-5-nitrobenzonitrile (415 mg, 2.5 mmol, 1 equiv) was added inone portion, and the reaction mixture was stirred for 10 minutes at 0°C. and allowed to warm up to ambient temperature over 1 hour. Thereaction mixture was concentrated, and the residue was taken in DCM andwater. The layers were separated, and the organic layer was washed withwater (×2), dried over Na₂SO₄, filtered, and concentrated to provide5-nitro-2-(oxetan-3-yloxy)benzonitrile (495 mg, 90%) as a brown-yellowsolid that was used without further purification.

¹H NMR (300 MHz; d₆-DMSO) δ 8.74-8.75 (m, 1H), 8.42-8.47 (m, 1H),7.09-7.12 (dd, 1H, J=0.9 Hz, J=9.3 Hz), 5.56-5.63 (m, 1H), 4.96-5.00 (m,2H), 4.59-4.63 (m, 2H); m/z=221 (M+H)⁺.

Following the general procedure described above, the following compoundswere prepared:

Preparation of 2-((R)-tetrahydrofuran-3-yloxy)-5-nitrobenzonitrile

(544 mg, 93%) from (R)-(−)-3-hydroxytetrahydrofuran. ¹H NMR (300 MHz;d₆-DMSO) δ 8.67-8.71 (m, 1H), 8.46-8.50 (m, 1H), 7.45-7.48 (d, 1H, J=9.6Hz), 5.33-5.40 (m, 1H), 3.74-3.95 (m, 4H), 2.27-2.40 (m, 1H), 1.89-2.06(m, 1H); m/z=235 (M+H)⁺.

Preparation of 2-((S)-Tetrahydrofuran-3-yloxy)-5-nitrobenzonitrile

(544 mg, 93%) from S)-(+)-3-hydroxytetrahydrofuran. ¹H NMR (300 MHz;d₆-DMSO) δ 8.67-8.71 (m, 1H), 8.46-8.50 (m, 1H), 7.45-7.48 (d, 1H, J=9.6Hz), 5.33-5.40 (m, 1H), 3.74-3.95 (m, 4H), 2.27-2.40 (m, 1H), 1.89-2.06(m, 1H); m/z=235 (M+H)⁺.

Preparation of 5-Nitro-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile

(510 mg, 82%) from 4-hydroxytetrahydropyran. ¹H NMR (300 MHz; d₆-DMSO) δ8.68-8.69 (d, 1H, J=3 Hz), 8.43-8.47 (m, 1H), 7.56-7.59 (d, 1H, J=9.3Hz), 4.97-5.05 (m, 1H), 3.82-3.87 (m, 2H), 3.50-3.60 (m, 2H), 1.99-2.06(m, 2H), 1.62-1.72 (m, 2H); m/z=248 (M+H)⁺.

Preparation of 5-Amino-2-(oxetan-3-yloxy)benzonitrile

General procedure: A round-bottom flask was charged with5-nitro-2-(oxetan-3-yloxy)benzonitrile (495 mg, 2.25 mmol), EtOH (20ml), and 10% Pd/C (50% in water, Degussa type E101; 100 mg, 20 wt % byweight of the starting nitro compound) giving a suspension. The flaskwas sealed with a rubber septum, degassed, and back-filled with H₂ (×3)from a balloon filled with H₂. The reaction mixture was stirred for 1hour using a H₂ filled balloon. The reaction mixture was filteredthrough a pad of Celite, and the pad of Celite was rinsed with MeOH. Thefiltrate was evaporated to dryness to provide5-amino-2-(oxetan-3-yloxy)benzonitrile (420 mg, 98%) as a pale yellowsolid that was used without further purification.

¹H NMR (300 MHz; d₆-DMSO) δ 6.77-6.81 (m, 2H), 6.53-6.60 (m, 1H),5.18-5.25 (1), 1H, J=6 Hz), 5.12 (bs, 2H), 4.84-4.89 (t, 2H, J=6.3 Hz),4.50-4.51 (t, 2H, J=6.2 Hz); m/z=191 (M+H)⁺.

Following the general procedure described above, the following compoundswere prepared:

Preparation of 2-((R)-Tetrahydrofuran-3-yloxy)-5-aminobenzonitrile

(465 mg, 98%) from 2-((R)-tetrahydrofuran-3-yloxy)-5-nitrobenzonitrile.¹H NMR (300 MHz; d₆-DMSO) δ 6.90-6.99 (m, 1H), 6.77-6.85 (m, 2H), 5.10(bs, 2H), 4.93-4.99 (m, 1H), 3.70-3.87 (m, 4H), 2.07-2.19 (m, 1H),1.89-1.96 (m, 1H); m/z=205 (M+H)⁺.

Preparation of 2-((S)-Tetrahydrofuran-3-yloxy)-5-aminobenzonitrile

(466 mg, 98%) from 2-((S)-tetrahydrofuran-3-yloxy)-5-nitrobenzonitrile.¹H NMR (300 MHz; d₆-DMSO) δ 6.90-6.99 (m, 1H), 6.77-6.85 (m, 2H), 5.10(bs, 2H), 4.93-4.99 (m, 1H), 3.70-3.87 (m, 4H), 2.07-2.19 (m, 1H),1.89-1.96 (m, 1H); m/z=205 (M+H)⁺.

Preparation of 5-Amino-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile

(361 mg, 81%) from 5-Nitro-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile.¹H NMR (300 MHz; d₆-DMSO) δ 7.00-7.03 (d, 1H, J=8.7 Hz), 6.73-6.84 (m,2H), 5.11 (bs, 2H), 4.42-4.48 (m, 1H), 3.79-3.85 (m, 2H), 3.40-3.47 (m,2H), 1.86-1.98 (m, 2H), 1.50-1.61 (m, 2H); m/z=219 (M+H)⁺.

Preparation of 4-fluoro-2-isopropoxy-1-nitrobenzene

A mixture of 5-fluoro-2-nitrophenol (50.0 g, 318 mmol, 1 equiv),2-iodopropane (96.0 mL, 955 mmol, 3 equiv), and K₂CO₃ (132.0 g, 955mmol, 3 equiv) in acetone (1 L) was heated to reflux for ON. Aftercooling to RT, the solid was filtered-off and rinsed with DCM, then thefiltrate was concentrated to dryness. The residue was taken in water,EtOAc, and 1N NaOH. The layers were separated, and the organic layer waswashed with 1N NaOH 1×. The organic layer was dried over Na₂SO₄,filtered, and concentrated to dryness to provide4-fluoro-2-isopropoxy-1-nitrobenzene (49.3 g, 78%) as a yellow-brownliquid that was used without further purification. ¹H NMR (300 MHz;d₆-DMSO) δ 7.91-7.96 (dd, 1H, J=6.3 Hz, 9.0 Hz), 7.31-7.35 (dd, 1H,J=2.4 Hz, 11 Hz), 6.88-6.95 (m, 1H), 4.77-4.89 (hep, 1H, J=6.0 Hz),1.27-1.29 (d, 6H, J=6.0 Hz); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −102.4(sextet); m/z=200 (M+H)⁺.

Preparation of 4-fluoro-2-isopropoxybenzenamine

A round-bottom flask was charged with4-fluoro-2-isopropoxy-1-nitrobenzene (49.3 g, 248 mmol), EtOH (600 mL),and 10% Pd/C (50% in water, Degussa type E101; 10 g, 20 wt % by weightof the starting nitro compound). The flask was sealed with a rubberseptum, degassed, and back-filled with H₂ (×3) from a balloon filledwith H₂. The reaction was stirred for 4 d using a H₂ filled balloon. Thereaction mixture was filtered through a pad of celite, and the pad ofcelite was rinsed with MeOH. The filtrate was evaporated to dryness toprovide 4-fluoro-2-isopropoxybenzenamine (41.0 g, 98%) as a brown liquidthat was used without further purification. ¹H NMR (300 MHz; d₆-DMSO) δ6.67-6.72 (dd, 1H, J=2.7 Hz, 11 Hz), 6.55-6.60 (dd, 1H, J=6.0 Hz, 8.7Hz), 6.43-6.49 (td, 1H, J=2.7 Hz, 8.4 Hz), 4.47-4.55 (m, 3H), 1.23-1.25(d, 6H, J=6.0 Hz); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −127.4 (sextet); m/z=170(M+H)⁺.

Preparation of 1-(4-fluoro-2-isopropoxyphenyl)-1H-tetrazol-5(4H)-one

Step 1: Phosgene (20% in toluene; 63 mL, 118.2 mmol, 2 equiv) was cooledto −10° C. (ice/MeOH) under argon. Then 4-fluoro-2-isopropoxybenzenamine(10.0 g, 59.1 mmol, 1 equiv) was added dropwise, and the reactionmixture was stirred at −10° C. for 15 min then heated to 90° C. for 3 hunder argon. After cooling, the reaction mixture was evaporated todryness, and the crude 4-fluoro-1-isocyanato-2-isopropoxybenzene wasdried in vacuo.

Step 2: This reaction with TMS-N₃ was performed behind a blast shield.The crude 4-fluoro-1-isocyanato-2-isopropoxybenzene was placed underargon and trimethylsilylazide (16 mL, 118.2 mmol, 2 equiv) was added.The reaction mixture was heated to refluxed for ON under argon. Aftercooling to RT, the reaction mixture was concentrated under vacuum, andthe residue was charged with saturated NaHCO₃ (100 mL) and stirredvigorously for 5-10 min, then the basic reaction mixture was dilutedwith EtOAc. The layers were separated, and the organic layer wasmonitored by TLC to confirm that all the product had been extracted bythe saturated NaHCO₃ solution. EtOAc was added to the combined basicaqueous extracts, and the pH was adjusted to <3 using 6N HCl. Theaqueous and organic layers were partitioned, and the aqueous layer wasextracted with EtOAc 3×. The combined organic layers were dried overNa₂SO₄, filtered, and the solvent removed under vacuum to provide1-(4-fluoro-2-isopropoxyphenyl)-1H-tetrazol-5(4H)-one (11.4 g, 81%) as abrown oil that was used without further purification.

¹H NMR (300 MHz; d₆-DMSO) δ 7.47-7.52 (dd, 1H, J=6.0 Hz, 8.4 Hz),7.19-7.24 (dd, 1H, J=2.7 Hz, 12 Hz), 6.87-6.94 (td, 1H, J=2.7 Hz, 8.1Hz), 4.63-4.75 (d, 6H, J=6.0 Hz), 1.17-1.19 (d, 6H, J=6.0 Hz); ¹⁹F NMR(282 MHz; d₆-DMSO) δ −107.2 (q); m/z=238 (M+H)⁺.

Preparation of1-(4-fluoro-2-isopropoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one

A mixture of 1-(4-fluoro-2-isopropoxyphenyl)-1H-tetrazol-5(4H)-one (4.10g, 17.2 mmol, 1 equiv), K₂CO₃ (7.15 g, 52.0 mmol, 3 equiv), andiodomethane (3.25 mL, 52.0 mmol, 3 equiv) in DMF (40 mL) was stirred atRT ON. The reaction mixture was partitioned between water and EtOAc,then the layers were separated. The aqueous layer was extracted withEtOAc 2×, and the combined organic layer was washed with brine 1×. Theorganic layer was dried over Na₂SO₄, filtered, and concentrated todryness to provide1-(4-fluoro-2-isopropoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one (3.50 g,81%) as a brown oil that was used without further purification. ¹H NMR(300 MHz; d₆-DMSO) δ 7.46-7.51 (dd, 1H, J=6.3 Hz, 8.4 Hz), 7.20-7.25(dd, 1H, J=2.7 Hz, 11 Hz), 6.87-6.94 (td, 1H, J=2.7 Hz, 8.1 Hz),4.63-4.75 (hept, 1H, J=6.0 Hz), 3.59 (s, 3H), 1.16-1.18 (d, 6H, J=6.0Hz); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −107.0 (sextet); m/z=253 (M+H)⁺.

Preparation of1-(4-fluoro-2-hydroxy-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one

A solution of1-(4-fluoro-2-isopropoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one (3.50 g,13.9 mmol, 1 equiv) in H₂SO₄ (35 mL) was cooled in ice/water bath. Tothe cooled solution, was added HNO₃ (fuming >90%; 715 uL, 15.3 mmol, 1.1equiv) dropwise, and the cooled reaction mixture was allowed to warm toRT over 2 h. The reaction mixture was quenched with ice, and extractedwith EtOAc 3×. The organic layer was then extracted with saturatedNaHCO₃ 3×. The basic aqueous layer was acidified to pH<3 with 6N HCl.Then, the acidic layer was extracted with EtOAc 3×, and the organiclayer was dried over Na₂SO₄, filtered, and concentrated to dryness toprovide1-(4-fluoro-2-hydroxy-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(2.94 g, 83%) as brown solid that was used without further purification.

¹H NMR (300 MHz; d₆-DMSO) δ 12.5 (bs, 1H), 8.41-8.44 (d, 1H, J=8.7 Hz),7.04-7.08 (d, 1H, J=13 Hz), 3.59 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−111.3 (q); m/z=256 (M+H)⁺.

Preparation of1-(5-amino-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one

A round-bottom flask was charged with1-(4-fluoro-2-hydroxy-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one (500mg, 1.96 mmol), EtOH (20 mL), HOAc (250 uL), and 10% Pd/C (50% in water,Degussa type E101; 100 mg, 20 wt % by weight of the starting nitrocompound). The flask was sealed with a rubber septum, degassed, andback-filled with H₂ (×3) from a balloon filled with H₂. The reaction wasstirred for 2 h using a H₂ filled balloon. The reaction mixture wasfiltered through a pad of celite, and the pad of celite was rinsed withMeOH. The filtrate was evaporated to dryness, and the product dried invacuo with a water bath at 45° C. to remove any residual HOAc to provide1-(5-amino-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one (437mg, 99%) as a brown solid that was used without further purification. ¹HNMR (300 MHz; d₆-DMSO) δ 9.46 (s, 1H), 6.67-6.72 (m, 2H), 4.80 (bs, 2H),3.56 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −128.6 (t); m/z=225 (M+H)⁺.

Example 30 Synthesis of1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 38)

To a microwave vial, was addedN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Racemic, single diastereomer; 100 mg, 0.335 mmol, 1 equiv),1-(5-amino-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Described in: WO2011/068898 120 mg, 0.455 mmol, 1.36 equiv), rac-BINAP(45 mg, 0.0726 mmol, 0.217 equiv), Cs₂CO₃ (327 mg, 1.00 mmol, 3 equiv),Pd(OAc)₂ (7 mg, 0.0291 mmol, 0.0870 equiv), and dioxane (4 ml). Themicrowave vial was capped and sonicated under vacuum for 5 minutes. Thereaction mixture was heated in the microwave at 120° C. for 2 hours. Thecooled reaction mixture was filtered using a pad of Celite and rinsedwith dioxane, and the filtrate was concentrated. The crude product waspurified by flash chromatography and eluted with DCM:2M NH₃/MeOH=100:0to 95:5 using 1% 2M NH₃/MeOH increments to provide the desired product1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 38) (Racemic, single diastereomer; 90 mg, 51%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.21 (s, 1H), 8.06 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.57-7.60 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.23-7.26 (m,1H), 6.80-6.83 (d, 1H, J=9.3 Hz), 5.34-5.27 (p, 1H, J=6.3 Hz), 4.79-4.84(t, 2H, J=6 Hz), 4.38-4.42 (t, 2H, J=5.7 Hz), 4.04 (bs, 1H), 3.62 (s,3H), 2.84 (bs, 1H), 1.89-2.42 (m, 4H), 1.13-1.74 (m, 5H), 1.06-1.13 (m,4H), 0.815 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −166.5 (s); m/z=526(M+H)⁺.

Example 31 Chiral HPLC resolution of1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compounds 39-40)

Chiral HPLC resolution of compound1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 38) (Racemic, single diastereomer, 55 mg) to enantiomers wasperformed.

Chiral HPLC method: Column: Chiralcel OJ-H, 4.6×250 mm, with guard.Mobil phase: 90% CO₂, 10% methanol w/0.1% triethylamine. Flow rate: 4ml/min. Injection volume: 10 μL. Concentration: approx 5 mg/ml.Detection: UV at 254 nm.

Compound 39

Compound 39: (>99% ee, 15 mg) has Rt=6.14 min.

¹H NMR (300 MHz; d₆-DMSO) δ 9.21 (s, 1H), 8.06 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.57-7.60 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.23-7.26 (m,1H), 6.80-6.83 (d, 1H, J=9.3 Hz), 5.34-5.27 (p, 1H, J=6.3 Hz), 4.79-4.84(t, 2H, J=6 Hz), 4.38-4.42 (t, 2H, J=5.7 Hz), 4.04 (bs, 1H), 3.62 (s,3H), 2.84 (bs, 1H), 1.89-2.42 (m, 4H), 1.13-1.74 (m, 5H), 1.06-1.13 (m,4H), 0.815 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −166.5 (s); m/z=526(M+H)⁺.

Compound 40

Compound 40: (>99% ee, 12 mg) has Rt=7.39 min.

¹H NMR (300 MHz; d₆-DMSO) δ 9.21 (s, 1H), 8.06 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.57-7.60 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.23-7.26 (m,1H), 6.80-6.83 (d, 1H, J=9.3 Hz), 5.34-5.27 (p, 1H, J=6.3 Hz), 4.79-4.84(t, 2H, J=6 Hz), 4.38-4.42 (t, 2H, J=5.7 Hz), 4.04 (bs, 1H), 3.62 (s,3H), 2.84 (bs, 1H), 1.89-2.42 (m, 4H), 1.13-1.74 (m, 5H), 1.06-1.13 (m,4H), 0.815 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −166.5 (s); m/z=526(M+H)⁺.

Example 32 Synthesis of5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile(Compound 41)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Racemic, single diastereomer; 150 mg, 0.502 mmol, 1 equiv),5-amino-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile (153 mg, 0.703 mmol,1.4 equiv), and PTSA monohydrate (95 mg, 0.502 mmol, 1 equiv) in IPA (5ml) were heated to 70° C. for 3 days. After cooling to ambienttemperature, the crude mixture was concentrated to dryness and taken inwater, EtOAc, and 1N NaOH. The layers were separated. The organic layerwas washed with 1N NaOH (×2), dried over Na₂SO₄, filtered, andconcentrated to dryness. The crude product was purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2MNH₃/MeOH increments to give compound5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)benzonitrile(Compound 41)(Racemic, single diastereomer; 182 mg, 76%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.11-8.12 (d, 1H, J=2.4 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.71-7.75 (dd, 2H, J=3 Hz, J=9.3 Hz),7.28-7.19 (m, 2H), 4.62-4.67 (m, 1H), 4.14 (bs, 1H), 3.80-3.87 (m, 2H),3.44-3.52 (m, 2H), 2.86 (bs, 1H), 2.31-2.55 (m, 1H), 1.08-2.06 (m, 15H),0.985 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −166.3 (s); m/z=481 (M+H)⁺.

Synthesis of Compounds with 2,2-Difluoro-5,5-dimethyloctahydroindolizine

Typical procedures for final compounds synthesis: an i-PrOH (2 ml)solution ofN-(2-chloro-5-fluoropyrimidin-4-yl)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-amine(33.5 mg, 0.1 mmol), aniline (0.14 mmol) and p-TSA.H₂O (28.5 mg, 0.15mmol) was stirred at 90° C. for 15-96 hours until <5% of chloro-SM wasdetected by LC-MS. Solvent was removed in vacuo and the product waspurified by RP-HPLC. Product was obtained as a formate salt and wasfree-based by following method: a MeOH solution of the salt was passedthrough a PL-HCO₃ column slowly, the column was further washed withMeOH, filtrate was collected and solvent was removed in vacuo to providefinal desired products.

Example 33 Synthesis of1-(5-(4-((7S,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 42)

¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J=7.8 Hz, 1H), 7.80 (d, J=3.1 Hz,1H), 7.06 (d, J=3.4 Hz, 1H), 6.87 (d, J=12.2 Hz, 1H), 5.08 (d, J=4.6 Hz,1H), 4.35-4.29 (m, 1H), 3.74 (s, 3H), 3.34 (ddd, J=14.3, 10.6, 6.9 Hz,1H), 3.01-2.92 (m, 1H), 2.81 (ddd, J=19.3, 15.3, 10.8 Hz, 1H), 2.42-2.28(m, 1H), 2.11-2.07 (m, 1H), 2.02-1.79 (m, 3H), 1.70 (dd, J=14.4, 4.5 Hz,1H), 1.59-1.49 (m, 1H, overlapped with water peak), 1.12 (s, 6H),0.87-0.81 (m, 2H), 0.60-0.55 (m, 2H); LRMS (M+H) m/z 548.34.

Example 34 Synthesis ofN²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-N⁴-((7S,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compound 43)

¹H NMR (300 MHz, CDCl₃) δ 8.90 (s, 1H), 8.73 (d, J=7.7 Hz, 1H), 7.80 (d,J=3.0 Hz, 1H), 7.10 (d, J=3.4 Hz, 1H), 6.90 (d, J=12.1 Hz, 1H), 5.13 (d,J=5.7 Hz, 1H), 4.31-4.24 (m, 1H), 3.34 (ddd, J=13.8, 10.7, 7.8 Hz, 1H),3.00-2.88 (m, 1H), 2.81 (ddd, J=19.7, 14.9, 10.8 Hz, 1H), 2.39-2.26 (m,1H), 2.08-1.80 (m, 4H), 1.69 (dd, J=14.4, 4.5 Hz, 1H), 1.62-1.49 (m, 1H,overlapped with water peak), 1.12 (s, 3H), 1.11 (s, 3H), 0.91-0.84 (m,2H), 0.64-0.58 (m, 2H); LRMS (M+H) m/z 518.33.

Example 35 5-Synthesis of(4-((7S,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-methylbenzonitrile(Compound 44)

¹H NMR (300 MHz, CDCl₃) δ 8.16 (d, J=2.4 Hz, 1H), 7.81 (d, J=3.1 Hz,1H), 7.41 (dd, J=8.4, 2.4 Hz, 1H), 7.20 (d, J=12.1 Hz, 1H), 6.88-6.86(m, 1H), 5.10 (d, J=5.6 Hz, 1H), 4.38-4.32 (m, 1H), 3.35 (ddd, J=13.8,10.7, 7.1 Hz, 1H), 3.04-2.94 (m, 1H), 2.83 (ddd, J=19.6, 15.1, 10.8 Hz,1H), 2.49 (s, 3H), 2.43-2.29 (m, 1H), 2.15 (br dd, J=13.6, 2.1 Hz, 1H),2.07-1.82 (m, 3H), 1.70 (ddd, J=13.6, 11.8, 4.3 Hz, 1H), 1.15 (s, 3H),1.14 (s, 3H); LRMS (M+H) m/z 431.26.

Example 36 Synthesis of1-(5-(4-((7R,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 45)

¹H NMR (300 MHz, CDCl₃) δ 8.53 (d, J=7.8 Hz, 1H), 7.79 (d, J=3.1 Hz,1H), 7.05 (d, J=3.1 Hz, 1H), 6.89 (d, J=12.2 Hz, 1H), 4.74 (d, J=6.5 Hz,1H), 4.17 (tdt, J=11.3, 7.3, 3.8 Hz, 1H), 3.72 (s, 3H), 3.30 (ddd,J=14.3, 10.7, 6.7 Hz, 1H), 2.85-2.68 (m, 2H), 2.40-2.26 (m, 2H),2.00-1.75 (m, 3H), 1.41 (dd, J=12.3, 12.3 Hz, 1H), 1.12 (s, 3H),1.17-1.06 (m, 1H), 0.98 (s, 3H), 0.85-0.79 (m, 2H), 0.59-0.53 (m, 2H);LRMS (M+H) m/z 548.35.

Example 37 Synthesis ofN²-(4-cyclopropyl-2-fluoro-5-(1H-tetrazol-1-yl)phenyl)-5-fluoro-N⁴-((7R,8aR)-2,2-difluoro-octahydro-5,5-dimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compound 46)

¹H NMR (300 MHz, CDCl₃) δ 8.89 (s, 1H), 8.67 (d, J=7.7 Hz, 1H), 7.78 (d,J=3.0 Hz, 1H), 7.07 (d, J=3.5 Hz, 1H), 6.90 (d, J=12.1 Hz, 1H), 4.77 (d,J=8.3 Hz, 1H), 4.14 (tdt, J=13.0, 8.8, 4.3 Hz, 1H), 3.28 (ddd, J=14.5,10.6, 5.8 Hz, 1H), 2.86-2.71 (m, 2H), 2.42-2.25 (m, 2H), 2.00-1.78 (m,2H), 1.56-1.47 (m, 1H, overlapped with water peak), 1.41 (dd, J=12.3,12.3 Hz, 1H), 1.14 (s, 3H), 1.18-1.06 (m, 1H), 0.95 (s, 3H), 0.89-0.83(m, 2H), 0.63-0.57 (m, 2H); LRMS (M+H) m/z 518.34.

Example 38 Synthesis of1-(2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 47)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(racemic, single diastereomer; 200 mg, 0.669 mmol, 1 equiv),1-(2-((R)-tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one(260 mg, 0.937 mmol, 1.4 equiv), and PTSA monohydrate (127 mg, 0.669mmol, 1 equiv) in IPA (7 ml) were heated to 70° C. for 3 days. Aftercooling to ambient temperature, the crude mixture was concentrated todryness and taken in water, EtOAc, and 1N NaOH. The layers wereseparated. The organic layer was washed with 1N NaOH (×2), dried overNa₂SO₄, filtered, and concentrated to dryness. The crude product waspurified by flash chromatography and eluted with DCM:2M NH₃/MeOH=100:0to 96:4 using 1% 2M NH₃/MeOH increments to give compound1-(2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 47) (Mixture of two diastereomers; 279 mg, 77%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.20 (s, 1H), 8.03 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.60-7.63 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.22-7.25 (d, 1H,J=7.8 Hz), 7.14-7.17 (d, 1H, J=9 Hz), 4.94-5.07 (m, 1H), 3.99-4.11 (m,1H), 3.79-3.83 (dd, 1H, J=4.5 Hz, J=9.9 Hz), 3.59-3.69 (m, 6H), 2.84(bs, 1H), 1.41-2.24 (m, 11H), 1.06-1.22 (m, 4H), 0.813 (s, 3H); ¹⁹F NMR(282 MHz; d₆-DMSO) δ −166.6 (s); m/z=540 (M+H)⁺.

Example 39 Synthesis of1-(2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 49)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Racemic, single diastereomer; 200 mg, 0.669 mmol, 1 equiv),1-(2-((S)-tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one(260 mg, 0.937 mmol, 1.4 equiv), and PTSA monohydrate (127 mg, 0.669mmol, 1 equiv) in IPA (7 ml) were heated to 70° C. for 3 days. Aftercooling to ambient temperature, the crude mixture was concentrated todryness and taken in water, EtOAc, and 1N NaOH. The layers wereseparated. The organic layer was washed with 1N NaOH (×2), dried overNa₂SO₄, filtered, and concentrated to dryness. The crude product waspurified by flash chromatography and eluted with DCM:2M NH₃/MeOH=100:0to 96:4 using 1% 2M NH₃/MeOH increments to give compound1-(2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 49) (Mixture of two diastereomers; 315 mg, 87%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.20 (s, 1H), 8.03 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.60-7.63 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.22-7.25 (d, 1H,J=7.8 Hz), 7.14-7.17 (d, 1H, J=9 Hz), 4.94-5.07 (m, 1H), 3.99-4.11 (m,1H), 3.78-3.83 (dd, 1H, J=4.5 Hz, J=9.9 Hz), 3.59-3.69 (m, 6H), 2.84(bs, 1H), 1.41-2.24 (m, 11H), 1.06-1.22 (m, 4H), 0.812 (s, 3H); ¹⁹F NMR(282 MHz; d₆-DMSO) δ −166.6 (s); m/z=540 (M+H)⁺.

Example 40 Synthesis of1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 51)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Racemic, single diastereomer; 200 mg, 0.669 mmol, 1 equiv),1-(5-amino-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Described in WO2011068898, 273 mg, 0.937 mmol, 1.4 equiv), and PTSAmonohydrate (127 mg, 0.669 mmol, 1 equiv) in IPA (7 ml) were heated to70° C. for 3 days. After cooling to ambient temperature, the crudemixture was concentrated to dryness and taken in water, EtOAc, and 1NNaOH. The layers were separated. The organic layer was washed with 1NNaOH (×2), dried over Na₂SO₄, filtered, and concentrated to dryness. Thecrude product was purified by flash chromatography and eluted withDCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2M NH₃/MeOH increments to givecompound1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 51) (racemic, single diastereomer; 321 mg, 87%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.19 (s, 1H), 8.06 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.57-7.61 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.22-7.25 (d, 1H,J=7.8 Hz), 7.18-7.21 (d, 1H, J=9 Hz), 4.44-4.57 (m, 1H), 3.97-4.12 (m,1H), 3.61-3.67 (m, 7H), 3.37-3.44 (m, 2H), 2.84 (bs, 1H), 1.42-2.24 (m,11H), 1.06-1.22 (m, 4H), 0.812 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.7 (s); m/z=554 (M+H)⁺.

Example 41 Synthesis of2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compound 57)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(racemic, single diastereomer; 200 mg, 0.669 mmol, 1 equiv),2-((R)-tetrahydrofuran-3-yloxy)-5-aminobenzonitrile (191 mg, 0.937 mmol,1.4 equiv), and PTSA monohydrate (127 mg, 0.669 mmol, 1 equiv) in IPA (7ml) were heated to 70° C. for 3 days. After cooling to ambienttemperature, the crude mixture was concentrated to dryness and taken inwater, EtOAc, and 1N NaOH. The layers were separated. The organic layerwas washed with 1N NaOH (×2), dried over Na₂SO₄, filtered, andconcentrated to dryness. The crude product was purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2MNH₃/MeOH increments to give compound2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compound 57) (two diastereomers; 295 mg, 95%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.10-8.11 (d, 1H, J=2.7 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.75-7.78 (dd, 1H, J=2.1 Hz, J=8.7 Hz),7.25-7.28 (d, 1H, J=7.8 Hz), 7.09-7.12 (d, 1H, J=9 Hz), 5.02-5.11 (m,1H), 4.11-4.21 (m, 1H), 3.71-3.91 (m, 4H), 2.85 (bs, 1H), 1.41-2.42 (m,11H), 1.08-1.27 (m, 4H), 0.987 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.3 (s); m/z=467 (M+H)⁺.

Example 42 Chiral HPLC separation of2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compounds 58-59)

Chiral HPLC separation of compound2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compound 57)(two diastereomers 55 mg) to diastereomers (each a singleenantiomer) was performed.

Chiral HPLC method: Column: Chiralcel OJ-H, 4.6×250 mm, with guard.Mobil phase: 90% CO₂, 10% methanol w/0.1% triethylamine. Flow rate: 4ml/min. Injection volume: 10 μL. Concentration: approx 5 mg/ml.Detection: UV at 254 nm.

Compound 58

Compound 58: (>99% ee, 16 mg) has Rt=3.90 min.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.10-8.11 (d, 1H, J=2.7 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.75-7.78 (dd, 1H, J=2.1 Hz, J=8.7 Hz),7.25-7.28 (d, 1H, J=7.8 Hz), 7.09-7.12 (d, 1H, J=9 Hz), 5.02-5.11 (m,1H), 4.11-4.21 (m, 1H), 3.71-3.91 (m, 4H), 2.85 (bs, 1H), 1.41-2.42 (m,11H), 1.08-1.27 (m, 4H), 0.987 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.3 (s); m/z=467 (M+H)⁺.

Compound 59

Compound 59: (>99% ee, 16 mg) has Rt=4.95 min.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.10-8.11 (d, 1H, J=2.7 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.75-7.78 (dd, 1H, J=2.1 Hz, J=8.7 Hz),7.25-7.28 (d, 1H, J=7.8 Hz), 7.09-7.12 (d, 1H, J=9 Hz), 5.02-5.11 (m,1H), 4.11-4.21 (m, 1H), 3.71-3.91 (m, 4H), 2.85 (bs, 1H), 1.41-2.42 (m,11H), 1.08-1.27 (m, 4H), 0.987 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.3 (s); m/z=467 (M+H)⁺

Example 43 Synthesis of2-((S-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compound 60)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Racemic, single diastereomer; 200 mg, 0.669 mmol, 1 equiv),2-((S)-tetrahydrofuran-3-yloxy)-5-aminobenzonitrile (191 mg, 0.937 mmol,1.4 equiv), and PTSA monohydrate (127 mg, 0.669 mmol, 1 equiv) in IPA (7ml) were heated to 70° C. for 3 days. After cooling to ambienttemperature, the crude mixture was concentrated to dryness and taken inwater, EtOAc, and 1N NaOH. The layers were separated. The organic layerwas washed with 1N NaOH (×2), dried over Na₂SO₄, filtered, andconcentrated to dryness. The crude product was purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2MNH₃/MeOH increments to give compound2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compound 60) (two diastereomers; 294 mg, 95%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.10-8.11 (d, 1H, J=2.7 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.75-7.78 (dd, 1H, J=2.1 Hz, J=8.7 Hz),7.25-7.28 (d, 1H, J=7.8 Hz), 7.09-7.12 (d, 1H, J=9 Hz), 5.02-5.11 (m,1H), 4.11-4.21 (m, 1H), 3.72-3.91 (m, 4H), 2.84 (bs, 1H), 1.40-2.42 (m,11H), 1.08-1.27 (m, 4H), 0.982 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.3 (s); m/z=467 (M+H)⁺.

Example 44 HPLC separation of compound 602-((S-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compounds 61-62)

HPLC separation of compound2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)benzonitrile(Compound 60) (mixture of two diastereomers 55 mg) to constituent singlediastereomers was performed.

HPLC method: Column: Chiralcel OJ-H, 4.6×250 mm, with guard. Mobilphase: 90% CO₂, 10% methanol w/0.1% triethylamine. Flow rate: 4 ml/min.Injection volume: 10 μL. Concentration: approx 5 mg/ml. Detection: UV at254 nm.

Compound 61

Compound 61: (>96% de, 17 mg) has Rt=6.01 min.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.10-8.11 (d, 1H, J=2.7 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.75-7.78 (dd, 1H, J=2.1 Hz, J=8.7 Hz),7.25-7.28 (d, 1H, J=7.8 Hz), 7.09-7.12 (d, 1H, J=9 Hz), 5.02-5.11 (m,1H), 4.11-4.21 (m, 1H), 3.71-3.91 (m, 4H), 2.85 (bs, 1H), 1.41-2.42 (m,11H), 1.08-1.27 (m, 4H), 0.987 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.3 (s); m/z=467 (M+H)⁺.

Compound 62

Compound 62: (>97% de, 18 mg) has Rt=6.90 min.

¹H NMR (300 MHz; d₆-DMSO) δ 9.14 (s, 1H), 8.10-8.11 (d, 1H, J=2.7 Hz),7.84-7.86 (d, 1H, J=3.9 Hz), 7.75-7.78 (dd, 1H, J=2.1 Hz, J=8.7 Hz),7.25-7.28 (d, 1H, J=7.8 Hz), 7.09-7.12 (d, 1H, J=9 Hz), 5.02-5.11 (m,1H), 4.11-4.21 (m, 1H), 3.71-3.91 (m, 4H), 2.85 (bs, 1H), 1.41-2.42 (m,11H), 1.08-1.27 (m, 4H), 0.987 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.3 (s); m/z=467 (M+H)⁺.

Example 45 Synthesis of5-(5-Fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)benzonitrile(Compound 63)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(racemic, single diastereomer; 200 mg, 0.669 mmol, 1 equiv),5-amino-2-(oxetan-3-yloxy)benzonitrile (178 mg, 0.937 mmol, 1.4 equiv),and PTSA monohydrate (127 mg, 0.669 mmol, 1 equiv) in IPA (7 ml) wereheated to 70° C. for 3 days. After cooling to ambient temperature, thecrude mixture was concentrated to dryness and taken in water, EtOAc, and1N NaOH. The layers were separated. The organic layer was washed with 1NNaOH (×2), dried over Na₂SO₄, filtered, and concentrated to dryness. Thecrude product was purified by flash chromatography and eluted withDCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2M NH₃/MeOH increments to givecompound5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(oxetan-3-yloxy)benzonitrile(Compound 63)(Racemic, single enantiomer; 163 mg, 54%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.16 (s, 1H), 8.14 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.71-7.74 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.23-7.26 (m,1H), 6.74-6.77 (d, 1H, J=9.3 Hz), 5.24-5.39 (p, 1H, J=6.3 Hz), 4.88-4.94(t, 2H, J=6 Hz), 4.49-4.58 (t, 2H, J=5.7 Hz), 4.15 (bs, 1H), 2.84 (bs,1H), 1.42-2.42 (m, 9H), 1.06-1.28 (m, 4H), 0.974 (s, 3H); ¹⁹F NMR (282MHz; d₆-DMSO) δ −166.2 (s); m/z=453 (M+H)⁺.

Example 46 Synthesis of1-(2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 48)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(single enantiomer; 35 mg, 0.116 mmol, 1 equiv),1-(2-((R)-tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one(45 mg, 0.162 mmol, 1.4 equiv), and PTSA monohydrate (22 mg, 0.116 mmol,1 equiv) in IPA (1.5 ml) were heated to 70° C. for 3 days. After coolingto ambient temperature, the crude mixture was concentrated to drynessand taken in water, EtOAc, and 1N NaOH. The layers were separated. Theorganic layer was washed with 1N NaOH (×2), dried over Na₂SO₄, filtered,and concentrated to dryness. The crude product was purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2MNH₃/MeOH increments to give compound1-(2-((R)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 48) (49 mg, 78%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.20 (s, 1H), 8.03 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.60-7.63 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.22-7.25 (d, 1H,J=7.8 Hz), 7.14-7.17 (d, 1H, J=9 Hz), 4.94-5.07 (m, 1H), 3.99-4.11 (m,1H), 3.79-3.83 (dd, 1H, J=4.5 Hz, J=9.9 Hz), 3.59-3.69 (m, 6H), 2.84(bs, 1H), 1.41-2.24 (m, 11H), 1.06-1.22 (m, 4H), 0.813 (s, 3H); ¹⁹F NMR(282 MHz; d₆-DMSO) δ −166.6 (s); m/z=540 (M+H)⁺.

Example 47 Synthesis of1-(2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 50)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Single diastereomer, single enantiomer; 53 mg, 0.178 mmol, 1 equiv),1-(2-((S)-tetrahydrofuran-3-yloxy)-5-aminophenyl)-4-methyl-1H-tetrazol-5(4H)-one(69 mg, 0.249 mmol, 1.4 equiv), and PTSA monohydrate (34 mg, 0.178 mmol,1 equiv) in IPA (2 ml) were heated to 70° C. for 3 days. After coolingto ambient temperature, the crude mixture was concentrated to drynessand taken in water, EtOAc, and 1N NaOH. The layers were separated. Theorganic layer was washed with 1N NaOH (×2), dried over Na₂SO₄, filtered,and concentrated to dryness. The crude product was purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2MNH₃/MeOH increments to give compound1-(2-((S)-tetrahydrofuran-3-yloxy)-5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 50) (75 mg, 78%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.20 (s, 1H), 8.03 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.60-7.63 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.22-7.25 (d, 1H,J=7.8 Hz), 7.14-7.17 (d, 1H, J=9 Hz), 4.94-5.07 (m, 1H), 3.99-4.11 (m,1H), 3.78-3.83 (dd, 1H, J=4.5 Hz, J=9.9 Hz), 3.59-3.69 (m, 6H), 2.84(bs, 1H), 1.41-2.24 (m, 11H), 1.06-1.22 (m, 4H), 0.812 (s, 3H); ¹⁹F NMR(282 MHz; d₆-DMSO) δ −166.6 (s); m/z=540 (M+H)⁺.

Example 48 Synthesis of1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 52)

A mixture ofN-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(Single diastereomer, single enantiomer; 53 mg, 0.178 mmol, 1 equiv),1-(5-amino-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Described in: WO 2011/068898, 73 mg, 0.249 mmol, 1.4 equiv), and PTSAmonohydrate (34 mg, 0.178 mmol, 1 equiv) in IPA (2 ml) were heated to70° C. for 3 days. After cooling to ambient temperature, the crudemixture was concentrated to dryness and taken in water, EtOAc, and 1NNaOH. The layers were separated. The organic layer was washed with 1NNaOH (×2), dried over Na₂SO₄, filtered, and concentrated to dryness. Thecrude product was purified by flash chromatography and eluted withDCM:2M NH₃/MeOH=100:0 to 96:4 using 1% 2M NH₃/MeOH increments to givecompound1-(5-(5-fluoro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-(tetrahydro-2H-pyran-4-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 52) (48 mg, 50%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.19 (s, 1H), 8.06 (s, 1H), 7.83-7.84 (d,1H, J=3.9 Hz), 7.57-7.61 (dd, 1H, J=2.1 Hz, J=8.7 Hz), 7.22-7.25 (d, 1H,J=7.8 Hz), 7.18-7.21 (d, 1H, J=9 Hz), 4.44-4.57 (m, 1H), 3.97-4.12 (m,1H), 3.61-3.67 (m, 7H), 3.37-3.44 (m, 2H), 2.84 (bs, 1H), 1.42-2.24 (m,11H), 1.06-1.22 (m, 4H), 0.812 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−166.7 (s); m/z=554 (M+H)⁺.

Example 49 Synthesis of1-(5-(4-((2R,7R,8aR)-2-fluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 64)

¹H NMR (300 MHz, CDCl₃) δ 8.57 (d, J=7.8 Hz, 1H), 7.78 (s, 1H), 7.09 (s,1H), 6.89 (d, J=12.2 Hz, 1H), 5.18 (dt, J=11.9, 5.7 Hz, 1H), 4.75 (d,J=8.2 Hz, 1H), 4.28-4.12 (m, 1H), 3.73 (s, 3H), 3.52-3.35 (m, 1H), 2.78(t, J=13.3 Hz, 1H), 2.71-2.52 (m, 1H), 2.33 (d, J=11.8 Hz, 1H),2.19-1.99 (m, 1H), 1.89-1.75 (m, 2H), 1.74-1.48 (m, 3H), 1.30 (t, J=12.2Hz, 1H), 1.15 (s, 3H), 1.00 (s, 3H), 0.87-0.78 (m, 2H), 0.61-0.51 (m,2H).

¹⁹F NMR (282 MHz, CDCl3) δ −129.42 (s), −167.50 (s), −168.35-−169.04(m).

LCMS (m/z): 530.5 (MH⁺).

Example 50 Synthesis of1-(5-(4-((2R,7S,8aR)-2-fluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 65)

¹H NMR (300 MHz, CDCl₃) δ 8.59 (d, J=7.8 Hz, 1H), 7.79 (d, J=3.1 Hz,1H), 7.10 (d, J=3.2 Hz, 1H), 6.86 (d, J=12.2 Hz, 1H), 5.30-5.04 (m, 2H),4.32 (s, 1H), 3.72 (s, 3H), 3.55-3.37 (m, 1H), 3.01-2.86 (m, 1H),2.80-2.60 (m, 1H), 2.08 (d, J=13.6 Hz, 2H), 1.92-1.75 (m, 3H), 1.63-1.37(m, 2H), 1.13 (s, 3H), 1.12 (s, 3H), 0.87-0.79 (m, 2H), 0.60-0.53 (m,2H).

¹⁹F NMR (282 MHz, CDCl₃) δ −129.67 (s), −167.84 (s), −168.37-−168.79(m).

LCMS (m/z): 530.5 (MH⁺).

Example 51 Synthesis of1-(5-(4-((2S,7R,8aR)-2-fluoro-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 67)

¹H NMR (300 MHz, CDCl₃) δ 8.54 (d, J=7.8 Hz, 1H), 7.78 (s, 1H), 7.05 (s,1H), 6.88 (d, J=12.2 Hz, 1H), 5.23-5.00 (m, 1H), 4.77 (d, J=8.0 Hz, 1H),4.19-4.08 (m, 1H), 3.71 (s, 3H), 3.18 (dd, J=22.9, 11.3 Hz, 1H),2.46-2.27 (m, 4H), 1.84-1.64 (m, 4H), 1.48 (t, J=12.3 Hz, 1H), 1.26-1.15(m, 3H), 0.93 (s, 3H), 0.86-0.78 (m, 2H), 0.59-0.53 (m, 2H).

¹⁹F NMR (282 MHz, CDCl₃) δ −129.23 (s), −164.71-−165.30 (m), −167.34(s).

LCMS (m/z): 530.5 (MH⁺).

Example 52 Synthesis of1-(5-(4-((2R,7R,8aR)-2-hydroxy-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 68)

¹H NMR (300 MHz, CDCl₃) δ 8.56 (d, J=7.7 Hz, 1H), 7.78 (s, 1H), 7.12 (s,1H), 6.90 (d, J=12.2 Hz, 1H), 4.79 (d, J=8.0 Hz, 1H), 4.40 (s, 1H),4.21-4.07 (m, 1H), 3.76 (s, 3H), 3.49 (s, 1H), 3.41 (dd, J=9.9, 6.8 Hz,1H), 2.70 (dd, J=16.6, 10.3 Hz, 1H), 2.32 (dd, J=9.8, 4.0 Hz, 2H), 2.08(s, 1H), 1.85-1.63 (m, 4H), 1.35 (t, J=12.3 Hz, 1H), 1.16 (s, 3H),1.01-0.88 (m, 3H), 0.81 (d, J=8.3 Hz, 2H), 0.57 (d, J=4.0 Hz, 2H).

LCMS (m/z): 528.4 (MH⁺).

Example 53 Synthesis of5-Fluoro-N2-(4-fluoro-3-(5-methyl-1H-tetrazol-1-yl)phenyl)-N4-(octahydro-5,5,8-trimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compounds 69-70)

Synthesis of Compounds 69 and 70

The mono-S_(N)Ar product (50.0 mg, 0.159 mmol) was dissolved in 4 ml ofisopropyl alcohol in a 2 dram vial. PTSA-monohydrate (45.3 mg, 0.238mmol) and the desired aniline (1.5 eq) were added and the vial wassealed. After heating over night at 105° C. using an anodized aluminumheating block the solvent was boiled off by removing the cap. Theproduct was isolated by preparative HPLC eluting with anacetonitrile/water gradient. Analysis by NMR showed that the isolatedproduct is a single diastereomer of unknown configuration.

Synthesis of5-Fluoro-N2-(4-fluoro-3-(5-methyl-1H-tetrazol-1-yl)phenyl)-N4-(octahydro-5,5,8-trimethylindolizin-7-yl)pyrimidine-2,4-diamine(Compound 70)

¹H NMR (300 MHz, d6-DMSO) δ 8.05-7.88 (m, 1H), 7.87-7.71 (m, 1H),7.70-7.50 (m, 2H), 6.50 (s, 1H), 5.46 (s, 1H), 4.17 (s, 1H), 3.70 (s,1H), 3.02 (s, 1H), 2.61-2.50 (m, 4H), 2.44 (t, J=0.9 Hz, 1H), 2.23 (d,J=20.1 Hz, 2H), 2.14 (d, J=14.0 Hz, 1H), 1.89 (s, 2H), 1.43 (d, J=12.9Hz, 1H), 1.35 (s, 3H), 1.24 (s, 3H), 1.20 (d, J=23.5 Hz, 3H), 0.97 (d,J=6.8 Hz, 2H); MS (ES) 470 (M+H), 468 (M−H).

Synthesis of1-(5-(5-Fluoro-4-(octahydro-5,5,8-trimethylindolizin-7-ylamino)pyrimidin-2-ylamino)-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 69)

¹H NMR (300 MHz, CDCl₃) δ 8.45 (d, J=7.8 Hz, 1H), 8.36 (s, 1H),7.78-7.72 (s br, 1H), 7.21 (s, 1H), 6.81 (d, J=12.1 Hz, 1H), 5.95 (s br,2H), 5.18 (s, 1H), 4.34 (s, 1H), 3.67 (s, 3H), 3.13 (m, 1H), 2.84 (m,1H), 2.60-2.45 (m, 1H), 2.28 (s, 1H), 2.24-2.03 (m, 4H), 2.05-1.66 (m,3H), 1.39 (s, 3H), 1.27 (s, 3H), 0.98 (d, J=7.0 Hz, 3H), 0.78 (d, J=8.3Hz, 1H), 0.56-0.46 (m, 2H); MS (ES) 526 (M+H), 524 (M−H).

Example 54 Synthesis of1-(5-(4-((7R,8aS)-5,5-dimethyl-octahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-methoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 22)

A mixture of(7R,8aS)—N-(2-choro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(49 mg, 0.16 mmol, 1 equiv),1-(5-amino-4-fluoro-2-methoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one (59mg, 0.25 mmol, 1.5 equiv) and pTsOH.H₂O (25 mg, 0.13 mmol, 0.8 equiv) inisopropanol (1.5 ml) was heated at 100° C. overnight in a sealed vial.After allowing to cool to room temperature, the solvent was removed andthe residue was purified by HPLC to give the product as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 8.39 (s, 1H), 7.85 (d, J=8.1 Hz, 1H), 7.78(d, J=3.9 Hz, 1H), 7.25 (d, J=12.6 Hz, 1H), 7.18 (d, J=8.1 Hz, 1H),4.05-3.90 (m, 1H), 3.75 (s, 3H), 3.59 (s, 3H), 2.85-2.75 (m, 1H),2.27-2.15 (m, 2H), 1.93-1.89 (m, 1H), 1.70-1.50 (m, 3H), 1.41-1.32 (m,1H), 1.24-1.15 (m, 1H), 1.11-0.95 (m, 2H), 1.04 (s, 3H), 0.76 (s, 3H);¹⁹F NMR (282 MHz; d₆-DMSO) δ −115.9 (s), −166.9 (s); m/z=502.1 (M+H)⁺;m/z=500.4 (M−H)⁺.

Example 55 Synthesis of1-(5-(4-((7R,8aS)-5,5-dimethyl-octahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-isopropoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 23)

A mixture of(7R,8aS)—N-(2-choro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(52 mg, 0.18 mmol, 1 equiv),1-(5-amino-4-fluoro-2-methoxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one (94mg, 0.36 mmol, 2 equiv) and pTsOH.H₂O (27 mg, 0.14 mmol, 0.8 equiv) inisopropanol (1.5 ml) was heated at 100° C. overnight in a sealed vial.After allowing to cool to room temperature, the solvent was removed andthe residue was purified by HPLC to give the product as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 8.38 (s, 1H), 7.82 (d, J=8.4 Hz, 1H), 7.78(d, J=3.9 Hz, 1H), 7.26 (d, J=12.6 Hz, 1H), 7.20 (d, J=8.1 Hz, 1H), 4.59(q, J=6.0 Hz, 1H), 4.05-3.90 (m, 1H), 3.59 (s, 3H), 2.90-2.80 (m, 1H),2.30-2.20 (m, 2H), 1.95-1.91 (m, 1H), 1.72-1.52 (m, 3H), 1.43-1.35 (m,1H), 1.22-1.00 (m, 3H), 1.55 (d, J=6.0 Hz, 6H), 1.05 (s, 3H), 0.78 (s,3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −116.2 (s), −167.0 (s); m/z=530.2(M+H)⁺; m/z=528.5 (M−H)⁺.

Example 56 Synthesis of Compound 71

¹H NMR (300 MHz; d₆-DMSO) δ 8.76 (s, 1H), 8.42-8.39 (m, 1H), 7.86 (d,J=3.6 Hz, 1H), 7.58-7.53 (m, 1H), 7.41 (t, J=6.5 Hz, 1H), 7.27 (br. d,1H), 4.04 (m, 1H), 2.81 (m, 1H), 2.42-2.20 (m, 2H), 1.98-1.89 (m, 1H),1.74-1.48 (m, 4H), 1.43-1.35 (m, 1H), 1.30-1.11 (m, 2H), 1.04 (s, 3H),0.83 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −165.3 (s), −135.6 (s),−135.1 (s), −116.7 (s), −112.5 (m); m/z=500.25 (M+H)⁺; rt=4.13 min (HPLCconditions-Protocol 1).

Example 57 Synthesis of Compound 72

¹H NMR (300 MHz; d₆-DMSO) δ 9.66 (s, 1H), 8.17 (d, J=11.9 Hz, 1H),7.94-7.92 (m, 2H), 7.43 (br. d, J=7.4 Hz, 1H), 7.27 (d, J=8.8 Hz, 1H),4.24-4.10 (m, 1H), 2.90-2.79 (m, 1H), 2.36-2.23 (m, 1H), 2.05-2.10 (m,1H), 1.70-1.44 (m, 4H), 1.30-1.15 (m, 3H), 1.09 (s, 3H), 0.98 (s, 3H);¹⁹F NMR (282 MHz; d₆-DMSO) δ −164.3 (s), −136.7 (s), −136.1 (s), −113.7(m), −109.5 (s); m/z=500.37 (M+H)⁺; rt=4.57 min (HPLCconditions-Protocol 1).

Example 58 Synthesis of Compound 76 Synthesis of4-(R,S)-Octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-cyclopropyl-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-yl)phenylamino)pyrimidine-5-carbonitrile (Compound 76)

To a solution of(R,S)-2-chloro-4-(octahydro-5,5-dimethylindolizin-7-ylamino)pyridimine-5-carbonitrile(Example 7; 0.300 g, 1.0 mmol) in iPrOH (25 mL),1-(5-amino-2-cyclopropyl-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(0.34 g, 1.4 mmol) and p-TSA (0.20 g, 1.1 mmol) were added and thereaction mixture was heated to 75° C. for 4 h. LCMS analysis indicatedthe completion of reaction. Removal of volatiles and purification of thecrude by column chromatography gave the desired product as p-toluenesulfonic acid salt. The salt was dissolved in 50 mL EtOAc andpartitioned with aq. 2N NaOH solution. The organic layers were separatedand dried over Na₂SO₄. Removal of the solvents under vacuum gave thedesired product in 78% yield (0.40 g).

¹H NMR (DMSO d₆, 300 MHz) δ: 9.43 (s, 1H), 8.29 (s, 1H), 7.65 (d, J=7.2Hz, 1H), 7.42 (d, J=8.1 Hz, 1H), 7.02 (d, J=12.0 Hz, 1H), 4.02 (br. s,1H), 3.60 (s, 3H), 2.76-2.81 (m, 1H), 2.15-2.24 (m, 2H), 1.79-1.83 (m,1H), 1.50-1.68 (m, 4H), 1.34-1.43 (m, 2H), 1.07-1.22 (m, 5H), 1.01 (s,3H), 0.82-0.86 (m, 2H), 0.63-0.67 (m, 2H); LCMS (m/z): 519 (MH⁺).

Example 59 Synthesis of1-(2-((S)-2-hydroxypropoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 81)

1-(5-amino-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one wasprepared according to Example 29 above.N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amineand 1-(5-amino-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-onewere conjugated to form the phenol (compound 89). The phenol (4.61 g,9.45 mmol), (S)-1-iodopropan-2-ol (7.02 g, 37.7 mmol) (synthesizedaccording to U.S. Application Publication No. 2005/0054701) and Cs₂CO₃(7.68 g, 23.6 mmol) were placed in a pressure vessel. DMA(dimethylacetamide) (50 ml) was added and the vessel was sealed. Thepale grey suspension was heated to 100° C. and stirred at thistemperature for 15 hours. After cooling down to room temperature, thereaction mixture was analyzed by TLC and HPLC to check for completion.The DMA was distilled off and water (100 ml) was added to the crudereaction mixture. The crude reaction mixture was extracted with DCM(2×100 ml) and EtOAc (1×100 ml), filtered through Na₂SO₄ and solventswere evaporated under reduced pressure to yield 6.6 gram of crudeproduct. The crude product was re-dissolved in DCM and absorbed onsilica gel. Further purification was carried out with a CombiFlash Goldcolumn (spherical silica, 220 gram) eluted with DCM and MeOH (2M NH₃)(gradient 0% to 6%). The clean fractions were combined to yield 3.04gram (59%, off-white solid) of compound 81. Reverse phase HPLC and¹H-NMR indicated a substantially pure product. Analysis by chiral SFC(12 min method) showed that the product also contained ˜1.2% of theother diastereomer and two diastereomeric regioisomers (˜1.0% and˜3.4%).

¹H NMR (300 MHz, d6-DMSO) δ 8.41 (s, 1H), 7.88 (d, J=8.4 Hz, 1H), 7.80(d, J=3.8 Hz, 1H), 7.27 (d, J=12.5 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 4.74(d, J=4.1 Hz, 1H), 4.08-3.94 (m, 1H), 3.93-3.76 (m, 3H), 3.60 (s, 3H),3.32 (m, 1H), 2.90-2.75 (m, 1H), 2.30-2.11 (m, 2H), 1.94 (d, J=11.0 Hz,1H), 1.78-1.47 (m, 4H), 1.47-1.33 (m, 1H), 1.30-1.09 (m, 2H), 1.09-0.99(m, 6H), 0.77 (s, 3H); MS (ES) m/z 546 (M+H)⁺.

Example 60 Synthesis of1-(2-(2-hydroxyethoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 77)

The phenol (compound 89) (9.90 g, 20.3 mmol), 2-bromoethanol (10.2 g,81.2 mmol) and Cs₂CO₃ (16.5 g, 50.7 mmol) were placed in a pressurevessel. DMA (dimethylacetamide) (100 ml) was added and the vessel wassealed. The reaction mixture was heated to 80° C. and stirred at thistemperature for 16 hours. The reaction was then analyzed by TLC and HPLCto check for completion. The DMA was distilled off, water (150 ml) wasadded and the crude reaction mixture was extracted with DCM (3×150 ml).The organic layers were combined, filtered through Na₂SO₄ and solventswere evaporated under reduced pressure. The crude product was furtherpurified using CombiFlash chromatography (regular silica gel, 330 gram)eluted with DCM and MeOH (2M NH₃) (gradient 0% to 6%). The cleanfractions were combined to yield 6.40 gram (59%) of compound 77 in theform of an off-white solid. Slightly less pure factions were alsocombined to yield another 3.35 gram (31%) of the product.

¹H NMR (300 MHz, d6-DMSO) δ 8.39 (s, 1H), 7.89 (d, J=8.4 Hz, 1H), 7.79(d, J=3.8 Hz, 1H), 7.28 (d, J=12.5 Hz, 1H), 7.18 (d, J=7.9 Hz, 1H), 4.71(t, J=5.6 Hz, 1H), 4.03 (t, J=4.9 Hz, 2H), 3.97 (dd, J=7.8, 4.0 Hz, 1H),3.65-3.53 (m, 5H), 3.34 (m, 1H), 2.80 (dd, J=8.4, 5.4 Hz, 1H), 2.27-2.09(m, 2H), 2.05 (s, 1H), 1.92 (d, J=11.4 Hz, 1H), 1.68 (dd, J=11.1, 6.6Hz, 1H), 1.62-1.45 (m, 3H), 1.37 (t, J=12.2 Hz, 1H), 1.27-1.05 (m, 1H),1.03 (s, 3H), 0.75 (s, 3H); ¹⁹F NMR (300 MHz, d6-DMSO) δ −116.5, −166.8;MS (ES) m/z 532 (M+H)⁺.

Example 61 Synthesis of Compound 80 Preparation of(7R,8aS)—N-(5-aminocarbonyl-2-chloropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

A mixture of (7R,8aS)-octahydro-5,5-dimethylindolizin-7-amine (1.25 g)and 5-aminocarbonyl-2,4-dichloropyrimidine (2.14 g, 1.5 eq.) in iPrOH(50 mL) was stirred at −10° C. to rt for 3 d. The solid was filtered offand washed with methanol (3×10 mL). The filtrate was evaporated undervacuum and the residue was purified by CombiFlash chromatography onsilica gel (2N NH₃ in MeOH/CH₂Cl₂=0-30%) to give the desired product(1.3 g, 54% over 3 steps).

m/z=324.11 (M+H)⁺.

Preparation of(7R,8aS)—N-(2-chloro-5-cyanopyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine

To a mixture of(7R,8aS)—N-(5-aminocarbonyl-2-chloropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(1.3 g) and triethylamine (1.72 mL) in THF (50 mL) was addedtrifluoroacetic anhydride (1.33 mL) at −78° C. After stifling for 2 h,the reaction was quenched with ice (150 mL). The aqueous solution wasextracted with EtOAc (6×150 mL). The organic layers were evaporatedunder vacuum and the residue was purified by CombiFlash chromatographyon silica gel (2N NH₃ in MeOH/CH₂Cl₂=0-20%) to give the desired product(1.1 g, 89%).

¹H NMR (CDCl₃, 300 MHz): δ 8.29 (s, 1H), 5.41 (d, J=6.0 Hz, 1H),4.34-4.29 (m, 1H), 3.01-2.94 (m, 1H), 2.57-2.52 (m, 1H), 2.39 (q, J=8.7Hz, 1H), 2.27-2.20 (m, 1H), 1.94-1.62 (m, 5H), 1.48-1.35 (m, 2H), 1.21(s, 3H), 1.06 (s, 3H); m/z=306.06 (M+H)⁺.

Synthesis of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxy-2-methylpropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 80)

A mixture of1-(5-amino-2-(2-hydroxy-2-methylpropoxy)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(305 mg),(7R,8aS)—N-(2-chloro-5-cyanopyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(262 mg), and benzenesulfonic acid (160 mg) in IPA (2 mL) were heated to80° C. overnight. After cooling to ambient temperature, the crudemixture was concentrated to dryness. The crude product was absorbed ontosilica gel and purified by CombiFlash chromatography (2N NH₃ inMeOH/CH₂Cl₂=0-30%) to give desired product (386 mg, 80%).

¹H NMR (300 MHz; d₆-DMSO) δ 9.32 (bs, 1H), 8.26 (s, 1H), 7.68 (d, 1H,J=7.8 Hz), 7.38 (d, 1H, J=7.2 Hz), 7.27 (d, 1H, J=12.0 Hz), 4.57 (s,1H), 3.96 (br, 1H), 3.72 (s, 2H), 3.58 (s, 3H), 2.78 (m, 1H), 2.16 (m,2H), 1.82-1.42 (m, 6H), 1.22-1.01 (m, 5H), 0.67 (bs, 3H); m/z=567.10(M+H)⁺.

Example 62 Synthesis of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((S)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 79)

A mixture of1-(5-amino-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one (210mg),(7R,8aS)—N-(2-chloro-5-cyanopyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(220 mg), and benzenesulfonic acid (200 mg) in IPA (2 mL) were heated to80° C. for overnight. After cooling to ambient temperature, the crudemixture was concentrated to dryness. The crude product was absorbed ontosilica gel and purified by CombiFlash chromatography (2N NH₃ inMeOH/CH₂Cl₂=0-30%) to give compound1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-cyanopyrimidin-2-ylamino)-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one(540 mg with some benzenesulfonic acid).

m/z=495.08 (M+H)⁺.

A mixture of1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-cyanopyrimidin-2-ylamino)-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one(600 mg), (S)-1-iodopropan-2-ol (1.13 g) and cesium carbonate (1.19 g)in DMA (5 mL) were heated to 100° C. for overnight. The reaction mixturewas diluted with water (100 mL), and extracted with EtOAc (2×100 mL).The organic layers were evaporated and purified by CombiFlashchromatography (2N NH₃ in MeOH/CH₂Cl₂=0-30%) to give compound 79 (400mg, 78%).

¹H NMR (300 MHz; d₆-DMSO) δ 9.32 (bs, 1H), 8.26 (s, 1H), 7.64 (d, 1H,J=8.7 Hz), 7.37 (d, 1H, J=7.8 Hz), 7.30 (d, 1H, J=12.3 Hz), 4.74 (d, 1H,J=4.2 Hz), 3.90-3.81 (m, 5H), 3.58 (s, 3H), 2.78 (m, 1H), 2.17 (m, 2H),1.82-1.42 (m, 6H), 1.18-1.01 (m, 5H), 0.68 (bs, 3H); m/z=553.06 (M+H)⁺.

Example 63 Synthesis of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxyethoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 78)

A mixture of1-(5-amino-2-(2-(tert-butyldimethylsilyl)oxyethoxy)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(200 mg),(7R,8aS)—N-(2-chloro-5-cyanopyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-amine(120 mg), and benzenesulfonic acid (120 mg) in IPA (2 mL) was heated to80° C. overnight. After cooling to ambient temperature, the crudemixture was concentrated to dryness. The crude product was absorbed ontosilica gel and purified by CombiFlash chromatography (2N NH₃ inMeOH/CH₂Cl₂=0-20%) to give desired product with benzenesulfonic acid.The methanol solution of the salt was passed through an ion-exchangeresin column to remove acid to give the free base of compound 78 (120mg, 57%).

¹H NMR (300 MHz; d₆-DMSO) δ 9.33 (bs, 1H), 8.26 (s, 1H), 7.64 (d, 1H,J=7.5 Hz), 7.37 (d, 1H, J=7.5 Hz), 7.32 (d, 1H, J=12.3 Hz), 4.71 (t, 1H,J=5.4 Hz), 4.05 (t, 2H, J=4.8 Hz), 4.01 (br, 1H), 3.58 (m, 5H), 2.78 (m,1H), 2.17 (m, 2H), 1.79-1.42 (m, 6H), 1.16-1.08 (m, 2H), 1.01 (s, 3H),0.69 (bs, 3H); m/z=539.11 (M+H)⁺.

Example 64 Synthesis of1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-((R)-2-hydroxypropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 82)

Method 1:

A mixture of anilino-phenol (300 mg, 1.33 mmol, 1 equiv), (R)-propyleneoxide (387 mg, 6.66 mmol, 5.0 equiv), K₂CO₃ (221 mg, 1.60 mmol, 1.2equiv) and ^(n)Bu₄NBr (43 mg, 0.13 mmol, 0.1 equiv) in 6 mL of acetonewas heated in a sealed vessel at 70° C. for 16 h. The mixture was cooledto room temperature, filtered and washed with EtOAc (˜50 mL). Thefiltrate was concentrated and the resulting residue was purified bycolumn chromatography on silica gel using hexane/EtOAc (1:4) as eluentto give 280 mg (Y=74%) of product A and B (˜3:1). This mixture wasdirectly used in the next step.

¹H NMR (CDCl₃, 300 MHz) of mixture: δ 6.92 (d, J=8.1 Hz, 1H, compoundB), 6.86 (d, J=9.0 Hz, 1H, compound A), 6.80 (d, J=11.1 Hz, 1H, compoundB), 6.77 (d, J=12.0 Hz, 1H, compound A), 4.12-4.05 (m, 3H, compound A),3.85-3.70 (m, 3H, compound B), 3.71 (s, 3H, compound B), 3.70 (s, 3H,compound A), 2.24 (s, 3H, compound B), 2.18 (s, 3H, compound B), 1.21(d, J=6.3 Hz, 3H, compound B), 1.19 (d, J=6.3 Hz, 3H, compound A);m/z=283.93 (M+H)⁺.

A mixture of above A and B (98 mg, 0.35 mmol, 1.3 equiv), mono-SNArproduct (80 mg, 0.27 mmol, 1 equiv), Pd(OAc)₂ (6 mg, 0.03 mmol, 0.1equiv), (±)BINAP (34 mg, 0.05 mmol, 0.2 equiv) and Cs₂CO₃ (264 mg, 0.81mmol, 3 equiv) in 2 mL of dioxane was microwaved at 120° C. for 2 hours.The mixture was filtered and washed with EtOAc (10 mL). The filtrate wasconcentrated and the residue was purified by HPLC to give compound 82.

¹H NMR (DMSO-d₆, 300 MHz): δ 8.39 (s, 1H), 7.86 (d, J=8.1 Hz, 1H), 7.78(d, J=3.9 Hz, 1H), 7.25 (d, J=12.6 Hz, 1H), 7.18 (d, J=7.2 Hz, 1H),4.05-3.90 (m, 1H), 3.88-3.78 (m, 3H), 3.59 (s, 3H), 2.80 (td, J=8.4, 3.0Hz, 1H), 2.26-2.16 (m, 3H), 1.93-1.89 (m, 1H), 1.69-1.50 (m, 4H), 1.37(t, J=12.6 Hz, 1H), 1.19-1.14 (m, 1H), 1.03 (s, 3H), 1.01 (d, J=5.7 Hz,3H), 0.75 (s, 3H); ¹⁹F NMR (DMSO-d₆, 282 MHz): δ −116.18 (s), −166.94(s); m/z=546.05 (M+H)⁺.

Method 2:

A mixture of the starting phenol (1.8 g, 3.69 mmol, 1 equiv),(R)-propylene oxide (1.1 mL, 14.77 mmol, 4.0 equiv), K₂CO₃ (1.0 g, 7.38mmol, 2.0 equiv) and “Bu₄NBr (0.12 g, 0.37 mmol, 0.1 equiv) in 15 mL ofacetone was heated in a sealed vessel at 70° C. for 4 days. The mixturewas cooled to room temperature, filtered and washed with EtOAc (˜100mL). The filtrated was washed with brine and concentrated. The resultingresidue was purified by column chromatography on silica gel using DCM/2NNH₃ in MeOH (95:5) as eluent to give 1.4 g (Y=71%) of compound 82.

Example 65 Synthesis of Compound 86 Preparation of1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-ol

2-Methylpropane-1,2-diol (1.08 g, 12.0 mmol, 1.0 equiv) in DCM (40 mL)under argon was cooled to 0° C. TEA (2.50 mL, 18.0 mmol, 1.5 equiv),DMAP (75.0 mg, 0.600 mmol, 0.05 equiv), and TBSCl (2.00 g, 13.2 mmol,1.1 equiv) were added sequentially, and the reaction was allowed to warmup to ambient temperature overnight. Complete conversion of reactant toproduct was confirmed by TLC eluted with EtOAc and visualized withKMnO₄. The solvent was removed in vacuo, and the residue was partitionedbetween water and EtOAc. The aqueous layer was extracted with EtOAc 2×,and the combined organics were dried with Na₂SO₄, filtered, andconcentrated to provide1-(tertbutyldimethylsilyloxy)-2-methylpropan-2-ol (2.05 g, 84%) as alight brown oil that was used without further purification.

¹H NMR (300 MHz; d₆-DMSO) δ 3.46 (s, 1H), 3.31 (s, 2H), 1.08 (s, 6H),0.920 (s, 9H), 0.0710 (s, 6H).

Preparation of1-(2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one

1-(Tertbutyldimethylsilyloxy)-2-methylpropan-2-ol (1.80 g, 8.81 mmol,1.10 equiv) in THF (30 mL) under argon gas, was cooled to −78° C.Potassium tert-butoxide (1.165 g, 9.61 mmol, 1.20 equiv) was added inone portion, and the reaction mixture was stirred for 15 min at −78° C.Then a solution of1-(2-fluoro-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one (preparedaccording to WO2011068898, which is incorporated herein by reference;1.915 g, 8.00 mmol, 1.00 equiv) in THF (10 mL) was added slowly, and thereaction mixture was stirred for 30 min at −78° C. and allowed to warmup to ambient temperature over 1 h. The reaction mixture wasconcentrated, and the residue was taken in EtOAc and water. The layerswere separated, and the aqueous layer was extracted with EtOAc 2×. Theorganic layer was dried over Na₂SO₄, filtered, and concentrated. Thecrude product was absorbed onto silica gel and purified by flashchromatography and eluted with heptane:EtOAc=100:0 to 30% EtOAc using10% EtOAc increments to provide1-(2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(2.28 g, 67%) as a yellow oil.

¹H NMR (300 MHz; d₆-DMSO) δ 8.44-8.53 (m, 2H), 7.54-7.57 (d, J=9.3 Hz,1H), 4.04 (s, 2H), 3.63 (s, 3H), 1.23 (s, 6H), 0.770 (s, 9H), 0.00300(s, 6H); m/z=424 (M+H)⁺.

Preparation of1-(5-amino-2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one

A round-bottom flask was charged with1-(2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)-5-nitrophenyl)-4-methyl-1H-tetrazol-5(4H)-one(2.54 g, 6.00 mmol), EtOH (60 mL), and 10% Pd/C (50% in water, Degussatype E101; 510 mg, 20 wt % by weight of the starting nitro compound).The flask was sealed with a rubber septum, degassed, and back-filledwith H₂ (3×) from a balloon filled with H₂. The reaction was stirred for4 h using a H₂ filled balloon. The reaction mixture was filtered througha pad of celite, and the pad of celite was rinsed with EtOAc/MeOH. Thefiltrate was evaporated to dryness to provide1-(5-amino-2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(2.26 g, 96%) as a light brown solid that was used without furtherpurification.

¹H NMR (300 MHz; d₆-DMSO) δ 6.97-7.00 (d, J=9.0 Hz, 1H), 6.74-6.78 (m,1H), 6.64-6.68 (m, 1H), 5.04 (bs, 2H), 3.66 (s, 2H), 3.60 (s, 3H), 1.17(s, 6H), 0.803 (s, 9H), 0.00300 (s, 6H); m/z=394 (M+H)⁺.

Preparation of1-(2-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 86)

A mixture of(7R,8aS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-aminehydrochloride (775 mg, 2.31 mmol, 1.00 equiv),1-(5-amino-2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(1.00 g, 2.54 mmol, 1.10 equiv), and PTSA monohydrate (880 mg, 4.62mmol, 2.00 equiv) in IPA (80 mL) was heated to reflux for 3 d asmonitored by LCMS. After the first day, in the morning, LCMS indicated aratio of OTBS-protected product:product=1.5:1. Thus, more PTSAmonohydrate (220 mg, 1.16 mmol, 0.50 equiv) was added and refluxing wascontinued. The reaction was checked by LCMS in the afternoon, whichindicated a ratio of OTBS-protected product:product=1:1.2. Thus, morePTSA monohydrate (220 mg, 1.16 mmol, 0.50 equiv) was added and refluxingwas continued. On the second day, in the morning, LCMS indicated a ratioof OTBS-protected product:product=1:6.5. Thus, more PTSA monohydrate(220 mg, 1.16 mmol, 0.50 equiv) was added and refluxing was continued.The reaction was checked by LCMS in the afternoon, which indicated aratio of OTBS-protected product:product=1:9. Thus, more PTSA monohydrate(220 mg, 1.16 mmol, 0.50 equiv) was added and refluxing was continued.On the third day, LCMS indicated the reaction was completed. Aftercooling to ambient temperature, the crude mixture was concentrated todryness. The residue was taken in water, EtOAc, and aqueous 1N NaOH. Theaqueous and organic layers were partitioned, and the aqueous layer wasextracted with EtOAc 2×. The organic layer was dried (Na₂SO₄), filtered,and the solvent removed under vacuum. The crude product was purified byflash chromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 94:6 using1% 2M NH₃/MeOH increments to give compound1-(2-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(compound 86) (870 mg, 70%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.82 (s, 1H), 8.07 (s, 1H), 7.83-7.84 (d,J=3.6 Hz, 1H), 7.58-7.61 (m, 1H), 7.21-7.24 (m, 1H), 7.09-7.12 (d, J=9.3Hz, 1H), 4.51 (s, 1H), 4.05 (bs, 1H), 3.65 (s, 2H), 3.59 (s, 3H), 2.83(m, 1H), 0.803-2.26 (m, 22H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −166.7 (s);m/z=542 (M+H)⁺.

Example 66 Preparation of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-3-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 83)

A mixture of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-chloropyrimidine-5-carbonitrilehydrochloride (200 mg, 0.584 mmol, 1.00 equiv),1-(5-amino-2-(1-(tert-butyldimethylsilyloxy)-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(255 mg, 0.643 mmol, 1.10 equiv), and PTSA monohydrate (225 mg, 1.17mmol, 2.00 equiv) in IPA (20 mL) was heated at 70° C. overnight. Afterthe first day, in the morning, LCMS indicated a ratio of OTBS-protectedproduct:product=3:1. Thus, more PTSA monohydrate (56 mg, 0.292 mmol,0.50 equiv) was added and temperature was raised to reflux. The reactionwas checked by LCMS in the afternoon, which indicated a ratio ofOTBS-protected product:product=1.5:1. Thus, more PTSA monohydrate (56mg, 0.292 mmol, 0.50 equiv) was added and refluxing was continued. Onthe second day, in the morning, LCMS indicated a ratio of OTBS-protectedproduct:product=1:4.5. Thus, more PTSA monohydrate (112 mg, 0.585 mmol,1.00 equiv) was added and refluxing was continued. The reaction a waschecked by LCMS in the afternoon, which indicated a ratio ofOTBS-protected product:product=1:8. Thus, more PTSA monohydrate (56 mg,0.292 mmol, 0.50 equiv) was added and refluxing was continued. On thethird day, LCMS indicated the reaction was completed. After cooling toambient temperature, the crude mixture was concentrated to dryness. Theresidue was taken in water, EtOAc, and aqueous 1N NaOH. The aqueous andorganic layers were partitioned, and the aqueous layer was extractedwith EtOAc 2×. The organic layer was dried (Na₂SO₄), filtered, and thesolvent removed under vacuum. The crude product was purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 94:6 using 1% 2MNH₃/MeOH increments to give compound4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-3-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(compound 83) (252 mg, 79%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.56 (bs, 1H), 8.30 (s, 1H), 8.04 (bs, 1H),7.63-7.66 (m, 1H), 7.46 (bs, 1H), 7.16-7.19 (d, J=9.3 Hz, 1H), 4.56 (s,1H), 4.15 (bs, 1H), 3.68 (s, 2H), 3.59 (s, 3H), 2.81 (m, 1H), 0.760-2.26(m, 22H); m/z=549 (M+H)⁺.

Example 67 Synthesis of Compound 84 Preparation of ethyl2-(4-amino-5-fluoro-2-(4-methyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)phenoxy)-2-methylpropanoate

A mixture of1-(5-amino-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one(5.00 g, 22.2 mmol, 1.00 equiv), ethyl 2-bromo-2-methylpropanoate (6.50g, 4.94 mL, 33.3 mmol, 1.50 equiv), K₂CO₃ (6.15 g, 44.4 mmol, 2.00equiv), and tetra-n-butylammonium bromide (716 mg, 2.22 mmol, 0.10equiv) in acetone (220 mL) was heated at reflux overnight. After coolingto RT, the reaction mixture was concentrated to dryness. The crudeproduct was taken in excess water and EtOAc. The layers were separated,and the aqueous layer was extracted with EtOAc 2×. The organic layer wasdried over Na₂SO₄, filtered, and concentrated to dryness. The crudeproduct was absorbed onto silica gel and purified by flashchromatography and eluted with DCM:MeOH=100:0 to 98% MeOH using 1% MeOHincrements to provide ethyl2-(4-amino-5-fluoro-2-(4-methyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)phenoxy)-2-methylpropanoate(5.60 g, 74%) as a clear brown oil.

¹H NMR (300 MHz; d₆-DMSO) δ 6.81-6.82 (d, J=5.7 Hz, 1H), 6.76-6.79 (d,J=8.4 Hz, 1H), 5.26 (bs, 2H), 4.11-4.18 (q, J=7.2 Hz, 2H), 3.58 (s, 3H),1.31 (s, 6H), 1.16-1.21 (t, J=6.9 Hz, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−128.5 (q); m/z=340 (M+H)⁺.

Preparation of1-(5-amino-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one

A solution of ethyl2-(4-amino-5-fluoro-2-(4-methyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)phenoxy)-2-methylpropanoate(5.60 g, 16.5 mmol, 1.00 equiv) in Et₂O (155 mL) under argon was cooledto 0° C. The ice-cooled solution was charged with lithium borohydride(791 mg, 36.3 mmol, 2.20 equiv) followed by MeOH (1.540 mL), whichresulted in the formation of a free-flowing precipitate. The reactionwas stirred at 0° C. for 30 min, then the ice-bath was removed, and thereaction was stirred at ambient temperature for 1 h. At ambienttemperature, the reaction was slowly quenched with aqueous 1 N NaOH (100mL, 100 mmol, 6.00 equiv), which dissolved the free-flowing precipitate.The layers were separated, and the aqueous layer was extracted with DCM2×. The organic layer was dried over Na₂SO₄, filtered, and concentratedto dryness to give1-(5-amino-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(4.60 g, 94%) as a white solid that was used without furtherpurification.

¹H NMR (300 MHz; d₆-DMSO) δ 7.05-7.11 (dd, J=3.9 Hz, 12.6 Hz, 1H),6.73-6.77 (d, J=3.9 Hz, 9.3 Hz, 1H), 5.20 (bs, 2H), 4.81 (bs, 1H), 3.58(s, 3H), 3.21 (s, 2H), 0.965 (s, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−129.1 (q); m/z=298 (M+H)⁺.

Preparation of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 84)

To a microwave vial, was added4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-chloropyrimidine-5-carbonitrilehydrochloride (100 mg, 0.292 mmol, 1.00 equiv),1-(5-amino-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(130 mg, 0.438 mmol, 1.50 equiv), rac-BINAP (37 mg, 0.0584 mmol, 0.200equiv), Cs₂CO₃ (286 mg, 0.877 mmol, 3.00 equiv), Pd(OAc)₂ (7 mg, 0.0292mmol, 0.100 equiv), and dioxane (3 mL). The microwave vial was cappedand sonicated under vacuum for 5 min. The reaction mixture was heated inthe microwave at 120° C. for 3 h. The cooled reaction mixture wasfiltered using a pad of celite and rinsed with dioxane, and the filtratewas concentrated. The crude product was purified by flash chromatographyand eluted with DCM:2M NH₃/MeOH=100:0 to 95:5 using 1% 2M NH₃/MeOHincrements to provide4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(compound 84) (65 mg, 39%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.32 (bs, 1H), 8.27 (s, 1H), 7.66-7.69 (d,J=8.4 Hz, 1H), 7.37-7.41 (m, 1H), 7.25-7.29 (d, J=12 Hz, 1H), 4.57 (s,1H), 3.98 (bs, 1H), 3.73 (s, 2H), 3.59 (s, 3H), 2.79 (m, 1H), 0.692-2.21(m, 22H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −113.6 (s); m/z=567 (M+H)⁺.

Example 68 Synthesis of1-(2-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 85)

To a microwave vial, was added(7R,8aS)—N-(2-chloro-5-fluoropyrimidin-4-yl)-octahydro-5,5-dimethylindolizin-7-aminehydrochloride (100 mg, 0.298 mmol, 1.00 equiv),1-(5-amino-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(133 mg, 0.447 mmol, 1.50 equiv), rac-BINAP (38 mg, 0.0597 mmol, 0.200equiv), Cs₂CO₃ (292 mg, 0.895 mmol, 3.00 equiv), Pd(OAc)₂ (7 mg, 0.0298mmol, 0.100 equiv), and dioxane (3 mL). The microwave vial was cappedand sonicated under vacuum for 5 min. The reaction mixture was heated inthe microwave at 120° C. for 3 h. The cooled reaction mixture wasfiltered using a pad of celite and rinsed with dioxane, and the filtratewas concentrated. The crude product was purified by flash chromatographyand eluted with DCM:2M NH₃/MeOH=100:0 to 95:5 using 1% 2M NH₃/MeOHincrements to provide1-(2-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(compound 85) (85 mg, 51%) as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 8.47 (s, 1H), 7.94-7.97 (d, J=8.7 Hz, 1H),7.81-7.82 (d, J=3.6 Hz, 1H), 7.31-7.35 (d, J=12.3 Hz, 1H), 7.20-7.25 (m,1H), 4.91-4.95 (t, J=5.7 Hz, 1H), 3.98 (bs, 1H), 3.58 (s, 3H), 3.29 (s,2H), 2.81 (m, 1H), 0.767-2.21 (m, 22H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ−117.8 (s), −166.4 (s); m/z=560 (M+H)⁺.

Example 69 Synthesis of1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(2-hydroxy-2-methylpropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 87)

Into a vessel was weighed compound 89 (10.2 g, 20.9 mmol), K₂CO₃ (3.5 g,25.1 mmol) and tetra-butylammonium bromide (674 mg, 2.09 mmol). Acetone(100 mL) then isobutylene oxide (5.6 mL, 62.8 mmol) was added and thevessel sealed. The mixture was then heated to a bath temperature of 78°C. for 5 days. The mixture was allowed to cool, then filtered and thefilter cake was washed with acetone (2×30 mL). The filtrate wasconcentrated under vacuum and the residue was partitioned between EtOAc(100 mL) and H₂O (100 mL). The aqueous layer was extracted with EtOAc(2×75 mL) and the combined organic extracts were dried (Na₂SO₄),filtered and the solvent removed under vacuum to leave a crude residue.The residue was dry-loaded on to silica gel and then purified by columnchromatography on silica gel using EtOAc/2M NH₃ in MeOH (1:0 to 9:1 inincrements of 0.02 2M NH₃ in MeOH) to give compound 87 (8.0 g, 68%) as asolid. Super-critical fluid chromatography using a chiral stationaryphase indicated that the product material contained ˜1.2% of animpurity, which may correspond to a regio-isomeric product arising fromring-opening of the epoxide at the more hindered terminus. Also obtainedfrom the column were mixed fractions containing the desired product (˜2g).

¹H NMR (300 MHz; d₆-DMSO): δ 8.39 (br. s, 1H), 7.91 (d, J=8.4 Hz, 1H),7.79 (d, J=3.8 Hz, 1H), 7.24 (d, J=12.5 Hz, 1H), 7.18 (br. s, 1H), 4.54(s, 1H), 4.03-4.90 (m, 1H), 3.71 (s, 2H), 3.58 (s, 3H), 2.83 (br. t,J=8.7 Hz, 1H), 2.24-2.10 (m, 2H), 1.94 (m, 1H), 1.74-1.50 (m, 5H), 1.38(t, J=12.2 Hz, 1H), 1.24-1.11 (m, 1H), 1.03 (s, 9H), 0.75 (s, 3H); ¹⁹FNMR (287 MHz; d₆-DMSO): δ −166.9 (d), −116.2 (s); m/z=560.15 [M+H]⁺.

Example 70 Synthesis of1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 88)

A mixture of1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one(900 mg, 1.85 mmol, 1 equiv), 3-iodooxetane (1.02 g, 5.54 mmol, 3equiv), and Cs₂CO₃ (665 mg, 2.03 mmol, 1.1 equiv) in DMA was heated to70° C. overnight. After cooling to RT, the reaction mixture was dilutedwith excess water (200 mL) and EtOAc (200 mL). The layers wereseparated, and the aqueous layer extracted with EtOAc 3×. The combinedorganics were dried over Na₂SO₄, filtered, and concentrated to dryness.The crude product was absorbed onto silica gel and purified by flashchromatography and eluted with DCM:2M NH₃/MeOH=100:0 to 95:5 using 1% 2MNH₃/MeOH increments to give compound 88 as a solid.

¹H NMR (300 MHz; d₆-DMSO) δ 8.44 (s, 1H), 7.90-7.93 (d, 1H, J=8.7 Hz),7.78-7.79 (d, 1H, J=3.9 Hz), 7.19-7.21 (s, 1H, J=8.1 Hz), 6.95-6.99 (s,1H, J=12 Hz), 5.28-5.36 (p, 1H, J=5.7 Hz), 4.83-4.88 (m, 2H), 4.39-4.43(m, 2H), 3.91-4.09 (m, 1H), 3.61 (s, 3H), 2.75-2.99 (m, 1H), 0.974-2.25(m, 13H), 0.750 (bs, 3H); ¹⁹F NMR (282 MHz; d₆-DMSO) δ −115.8 (t),−166.8 (s); m/z=544 (M+H)⁺.

Example 71 Synthesis of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)-2-fluoro-4-hydroxyl-phenylamino)pyrimindine-5-carbonitrile

To a mixture of(7R,8aS)—N-(2-chloro-5-cyanopyrimidinal-4-yl)-octahydro-5,5dimethylindolizin-7-amine(150 mg, 1.0 eq.) in isopropyl alcohol (2.45 ml, 0.2M),5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)-2-fluoro-4-hydroxyl-phenyamine(144 mg, 1.3 eq.) was added, followed by p-toluenesulfonic acid (112 mg,1.2 eq.), and was heated at 80° C. overnight under nitrogen. Uponcompletion of the reaction indicated by LC/MS, the mixture was cooled toroom temperature, and concentrated under reduced pressure. The residuewas dissolved into ethyl acetate, absorbed into silica gel and purifiedby Combi-flash chromatography (2N NH₃ in MeOH/EtOAc=0-20%), yield 160 mgof desired product as a light yellow solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.24 (bs, 1H), 8.25 (s, 1H), 7.5 (d, J=7.5Hz, 1H), 7.33 bs, 1H), 6.87 (d, J=11.7 Hz, 1H), 3.57 (s, 3H), 2.82 (bs,1H), 1.96-1.67 (m, 8H), 1.13 (m, 6H), 0.76 (m, 2H); m/z=495.01 (M+H)⁺.

Synthesis of4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((R)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimindine-5-carbonitrile(Compound 90)

4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)-2-fluoro-4-hydroxyl-phenylamino)pyrimindine-5-carbonitrile(467 mg, 1.0 eq.) was added to N,N-dimethylacetamide (4.7 ml, 0.2M),followed by Cs₂CO₃ (922 mg, 3.0 eq.) and 3-iodo-2-(R)-propanol (0.88 g,5.0 eq.) and heated at 100° C. under nitrogen overnight. It was cooledto room temperature and concentrated under reduced pressure. The residuewas treated with ethyl acetate (10 ml) and treated with saturated NaHCO₃(10 ml). The aqueous layer was extracted with ethyl acetate (2×10 ml),and the combined organic solution was washed with brine (10 ml), anddried with Na₂SO₄. Solid was filtered off, and the organic solution wasconcentrated under reduced pressure. 5 ml of ethyl acetate was added tothe residue, silica gel was added to form a thick paste, and wasconcentrated under reduced pressure. This silica gel mixture was loadedonto the Combiflash chromatography and product was eluted using 2N NH₃in MeOH (0-20%)/Ethyl Acetate (100%-80%) gradient to yield 124 mg ofproduct as a light beige solid.

¹H NMR (300 MHz; d₆-DMSO) δ 9.33 (s, 1H), 8.26 (s, 1H), 7.65 (d, J=8.1Hz, 1H), 7.37 (d, J=7.5 Hz, 1H), 7.3 (d, J=12 Hz, 1H), 4.75 (d, J=4.2Hz, 1H), 3.97-3.8 (m, 3H), 3.58 (s, 3H), 2.76 (m, 1H), 2.20 (m, 2H),1.83-1.4 (m, 8H), 1.22-1.07 (m, 2H), 1.03 (m, 6H), 1.0 (bs, 1H);m/z=553.04 (M+H)⁺.

BIOLOGICAL EXAMPLES Example 72 PKC Assay

The inhibition of PKC-alpha, PKC-beta, PKC-delta, PKC epsilon andPKC-theta activity is determined via ELISA as follows: NUNC MAXISORP(#436110) or Costar High Binding (#3922) plates are coated with 0.01mg/ml Neutravidin (Pierce #PI-31000) in 1×PBS (100 μL/well) for 18-24hours at 4° C. When ready to be used, plates are washed with 1×PBST andthen blocked with 2% BSA in 1×PBST (100 μL/well) for a minimum of 1 hourat room temperature. The reactions are conducted in a volume of 60μL/well. When ready to begin, the plates are washed with 1×PBST toremove the 2% BSA blocking solution. Reaction solution containing thenecessary buffer components as well as the appropriate concentrations ofATP and peptide substrate is then added to each well (see Table 3).Appropriate concentrations of test compound is then added—with thevolume added should taking into consideration the DMSO tolerance of thekinases being about 0.2%. The reaction is then initiated by the additionof kinase—the approximate final concentration of which is listed inTable 3 (note that this will vary depending on the batch to batchvariability in the activity of enzymes). After allowing the reaction tostand at room temperature for 20 minutes, the plates are then washedwith 1×PBST.

TABLE 3 Buffer [ATP] Time Kinase components (uM) [peptide] (uM) (min) 1°and 2° antibodies Notes PKCs 20 mM 1 μM 1 μM PKC peptide 20 min RabbitpSer PKC 0.15 mg/ml DAG α: ~8 ng/ml  Hepes (biotin- substrate Ab (Cell(Sigma #D0138) β: ~16 ng/ml pH 7.4 RFARKGSLRQKNV) Signaling #2261); 0.75mg/ml δ: ~13 ng/ml 5 mM MgCl₂ (Invitrogen #P2760) HRP-goat a-rabbitPhosphoserine ε: ~13 ng/ml 0.2 mM (Jackson (Sigma #P6641) θ: ~8 ng/ml CaCl₂ Immunoresearch DMSO tolerance 1 mM DTT #111-035-003) ~0.2% 0.05%Chaps

After removal of the reaction mixture from the plate and washing with1×PBST, an antibody developing solution containing a 1:10,000 dilutionof the appropriate primary and secondary antibodies (Table 3) in a 0.1%BSA solution in 1×PBST is then added to each well (100 μL/well). This isthen allowed to stand at room temperature for a minimum of 1 hour. Afterthis time, the plates are once again washed with 1×PBST. The SuperSignalELISA Pico Chemiluminescent substrate (Pierce #PI-37069) is then added(100 μL/well) and the plate is read on a luminescence plate reader.

Example 73 PKC Assay

Alternatively, the inhibition of PKC activity is measured by monitoringthe production of phosphorylated peptide by fluorescence polarization atdifferent concentrations of the inhibitor. Reactions are carried out in96-well plate format with a total volume of 20 μL, containing 20 mMHEPES, pH 7.4, 5 mM MgCl₂, 0.2 mM CaCl₂, 1 mM DTT, 0.02% Brij-35, 0.1mg/ml phosphatidylserine, 0.02 mg/ml dioleoyl-sn-glycerol and 5 μM eachof ATP and the peptide substrate. Compounds are first diluted seriallyin DMSO and then transferred to a solution containing the aboveconcentrations of HEPES, MgCl₂, CaCl₂, DTT, and Brij-35 to yield 5×compound solutions in 2% DMSO, which is then added to the reactionsolution. Reactions are initiated by the addition of PKC at a typicalconcentration as described in Table 4, and then allowed to incubate atroom temperature for 20 minutes. At the end of this time, a combinationof quench (EDTA) and detection (peptide tracer and antibody) reagents isadded using the protocol of Invitrogen P2748. After a 30 minutes periodof incubation, the amount of phosphorylated peptide generated ismeasured by fluorescence polarization (Ex=485 nm, Em=535 nm) using aTecan Polarian instrument.

TABLE 4 Typical enzyme Peptide substrate SEQ ID Enzyme sourceconcentration PKC theta RFARKGSLRQKNV Seq ID No. 1 UpstateBiotechnologies, 40 ng/ml Temecula, CA, cat. #14- 444 PKC RFARKGSLRQKNVSeq ID No. 1 Upstate Biotechnologies, 50 ng/ml epsilon Temecula, CA,cat. #14- 518

Example 74 Calcium Influx

HEK-FLPTREX cells are stably transfected with pcDNA5/FRT/TO+hTRPV4a, ratTRPV1-HA or rTRPA1-HA are grown in Dulbecco's Modified Eagle's Medium(DMEM) containing 10% tetracycline-free fetal bovine serum, hygromycin(50 μg/ml) and blasticidin (10 μg/ml). Cells are treated withtetracycline (0.1 μg/ml, 20 h) to induce TRP expression. DRG fromthoracic and lumbar spinal cord of rats or mice are minced in coldHank's Balanced Salt Solution (HBSS) and incubated for 60 at 37° C. inDMEM containing 1 mg/ml of collagenase type IA and 0.1 mg/ml of DNAsetype IV, pelleted and incubated with 0.25% trypsin for 30 minutes.Neurons are pelleted, suspended in DMEM containing 10% fetal bovineserum, 10% horse serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, 2mM glutamine, dissociated by gentle trituration until the solutionappears cloudy and homogeneous and plated on glass coverslips coatedwith PolyOnitine/laminin. Neurons are cultured for 3-4 days before theexperiment.

Cells grown on coverslips or on a 96 multiwell plate are incubated inHBSS (pH 7.4) containing Ca2+ and Mg2+, 20 mM HEPES buffer, 0.1% BSA,100 U/ml penicillin, 100 μg/ml streptomycin, with 2.5-5 μM Fura-2AM(Invitrogen) for 20-45 minutes at 37° C. Cells are washed andfluorescence is measured at 340 nm and 380 nm excitation and 510 nmemission in a F-2500 spectrophotometer, or in a Flexstation 3 MicroplateReader III (for the measurement of the calcium in the cell population)or using a Zeiss Axiovert microscope, an ICCD video camera and a videomicroscopy acquisition program (for the measurement of the calciuminflux in the single neurons). Substances are injected directly into thechamber (20 ml into 2 ml, for the spectrophotometer; 20 ml in 200 ml forthe Flexstation, 50 ml in 350 ml in the chamber for the single cells).

Example 75 In Vivo Hyperplasia

Mechanical pain is quantified as the number of times the hind paw iswithdrawn in response to 5 applications of a 0.173 mN von Frey hair.Responses are expressed as a percentage (e.g. 3 withdrawals out of 5 arerecorded as 60%) and mechanical hyperalgesia defined as increase in thepercentage of withdrawal compared to basal measurement. 2) Mechanicalpain is quantified using the ‘up-down paradigm’, determining the 50%response threshold to the von Frey filaments applied to the mid-plantarsurface for 5 s or until a withdrawal response occurred. Von Freyfilaments are in this range of intensities: 1.65, 2.44, 2.83, 3.22,3.61, 3.84, 4.08.

Thermal hyperalgesia is assessed in mice using a plantar test apparatusand quantified as the latency of paw withdrawal to a radiant heat.Thermal hyperalgesia is defined as a decrease in the withdrawal latencycompared to the basal measurement. After measuring basal level mice,under light halothane anesthesia (5%), are injected with testingcompound into the left or right paws (5-10 μl intraplantar injection)and paw withdrawal measurements repeated at different time point. Toassess PAR2 TRPV1, TRPV4 and TRPA1 mediated hyperalgesia andpotentiation of TRPV-mediated responses, mice are treated with PAR2-APfor 15 minutes followed by capsaicin, 4αPDD or HNE. To assess the roleof protein kinases, the antagonists or the corresponding vehicles areinjected 20-30 minutes before the challenge with agonists. The effectsinduced by the different treatments are evaluated within the same ratcomparing the responses recorded in the right paw (receiving for examplesaline, or vehicle) with the responses obtained in the left paw(receiving for example PAR2-AP or 4αPDD).

Formalin induced hyperalgeisa is assessed using 5% solution of formalinadministered by intradermal injection into the dorsal surface of themouse or rat forepaw to induce a painful behavior. Pain is accessed on afour-level scale related to posture: 0, normal posture; 1, with theinjected paw remaining on the ground but not supporting the animal; 2,with the injected paw clearly raised; and 3, with the injected paw beinglicked, nibbled, or shaken. Animals are observed and scored for behaviorat 3 minutes after the injection (defined as initial phase that resultsfrom the direct stimulation of nociceptors), and then at 30-60 minutesafter the injection (defined as second phase that involves a period ofsensitization during which inflammatory phenomena occur). Thenociceptive behavioral score for each 3-minutes interval is calculatedas the weighted average of the number of seconds spent in each behavior.2.5% solution of formalin is administered by intraplantar injection andthermal and mechanical pain measured as described above after 30-60minutes. To assess the role of protein kinases, antagonists or theirvehicles (control) are injected into the right paws 20-30 minutes beforeformalin. Nociceptive behavior will be scored for each rats and comparedto control.

Exemplary compounds exhibited in vivo activity in one or more diseasemodels. For example Compound 17 had an ED50 of 30 mg/kg in bothpretreatment and treatment dosing in an PLP induced experimentalautoimmune encephalomyelitis (EAE) model in SJL mice. As is known tothose of skill in the art, EAE is considered to be a model of multiplesclerosis in humans. Other exemplary compounds demonstrated in vivoefficacy in rodents in contact dermatitis and/or adjuvant inducedarthritis models.

Example 76 IL-2 ELISA, Human Primary T Cell, Anti CD3+CD28+

Compounds 1-90 were tested in a whole cell functional assay for PKCmediated IL-2 production as follows. Human primary T cell isolation andculture: Human primary T cells were prepared as follows. Whole blood wasobtained from a healthy volunteer, mixed 1:1 with PBS, layered on toFicoll Hypaque (Amersham Pharmacia Biotech, Piscataway, N.J., Catalog#17-1440-03) in 2:1 blood/PBS:ficoll ratio and centrifuged for 30minutes at 4° C. at 1750 rpm. The cells at the serum: ficoll interfacewere recovered and washed twice with 5 volumes of PBS. These freshlyisolated human peripheral blood mononuclear cells were cultured inYssel's medium containing 40 U/ml IL2 in a flask pre-coated with 1 μg/mlαCD3 and 5 μg/ml αCD28 (Anti-Human CD3, BD Pharmingen Catalog #555336,Anti-Human CD28, Beckman Coulter Catalog #1M1376). The cells werestimulated for 3-4 days, then transferred to a fresh flask andmaintained in RPMI (RPMI-1640 with L-Glutamine; Mediatech, Inc., HerndonVa., cat. #10-040-CM) with 10% FBS and 40 U/ml IL-2. The primary T-cellswere then washed twice with PBS to remove the IL-2.

Primary T cell stimulation and IL2 ELISA: Human primary T cells (100,000cells per well) were pre-incubated with or without test compound inYssel's medium for 1 hour at 37° C. Cells were then stimulated bytransferring them to round-bottom 96-well plates pre-coated with 1 μg/mlαCD3 and 5 μg/ml αCD28. For counter assay, cells were instead stimulatedby adding 8× stock solutions of PMA and ionomycin in Yssels (for finalconcentrations of 0.5 ng/ml PMA and 0.1 uM ionomycin, both fromCalbiochem). Cells were incubated at 37° C. for 24 hours before 100 μLsupernatants were harvested for quantification of IL-2 by ELISA usingHuman IL-2 Duoset ELISA Kit from R and D Systems, Cat. # DY202E.Compounds 1-90 blocked IL-2 production with an EC50 of less than 10micromolar.

Table 5 shows the EC50 values of Compounds 1-90 according to the aboveassay. In Table 5, an EC50 of A is less than 0.1 μM, B<0.5 μM, C<1 μM,D<10 μM, and E>10 μM.

TABLE 5 Compound EC50 (μM)  1 B  2 A  3 C  4 D  5 B  6 B  7 D  8 C  9 B10 B 11 D 12 C 13 A 14 A 15 B 16 A 17 A 18 C 19 C 20 D 21 D 22 B  23) A24 B 25 D 26 D 27 D 28 A 29 D 30 E 31 D 32 D 33 D 34 D 35 D 36 D 37 D 38B 39 B 40 A 41 B 42 D 43 D 44 D 45 D 46 C 47 A 48 A 49 A 50 A 51 B 52 B53 A 54 A 55 B 56 A 57 B 58 D 59 B 60 B 61 C 62 B 63 B 64 B 65 D 66 A 67B 68 B 69 C 70 D 71 C 72 C 73 B 74 B 75 B 76 A 77 A 78 A 79 A 80 A 81 A82 A 83 A 84 B 85 B 86 A 87 A 88 B 89 D 90 A

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of inhibiting a protein kinase C (PKC)activity comprising contacting a PKC with a compound selected from:Compound 77:1-(2-(2-hydroxyethoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;Compound 78:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxyethoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;Compound 79:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(44-((S)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;Compound 80:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxy-2-methylpropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;Compound 81:1-(24-((S)-2-hydroxypropoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one;Compound 82:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-((R)-2-hydroxypropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;Compound 83:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-3-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;Compound 84:4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-2-(2-fluoro-4-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-methyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile;Compound 85:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;Compound 86:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;Compound 87:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(2-hydroxy-2-methylpropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;and Compound 90:4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((R)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimindine-5-carbonitrile.2. The method of claim 1, wherein the compound is1-(2-(2-hydroxyethoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one (Compound 77).
 3. The method of claim 1, wherein the compoundis4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxyethoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 78).
 4. The method of claim 1, wherein the compound is4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((S)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 79).
 5. The method of claim 1, wherein the compound is4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(2-hydroxy-2-methylpropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 80).
 6. The method of claim 1, wherein the compound is1-(2-((S)-2-hydroxypropoxy)-5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluorophenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 81).
 7. The method of claim 1, wherein the compound is1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-((R)-2-hydroxypropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 82).
 8. The method of claim 1, wherein the compound is4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-(1-hydroxy-2-methylpropan-2-yloxy)-3-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 83).
 9. The method of claim 1, wherein the compound is4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-2-(2-fluoro-4-(1-hydroxy-2-methylpropan-2-yloxy)-5-(4-methyl-5-oxo-4,5-dihydro-1H-tetrazol-1-yl)phenylamino)pyrimidine-5-carbonitrile(Compound 84).
 10. The method of claim 1, wherein the compound is1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 85).
 11. The method of claim 1, wherein the compound is1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-2-(1-hydroxy-2-methylpropan-2-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 86).
 12. The method of claim 1, wherein the compound is1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(2-hydroxy-2-methylpropoxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 87).
 13. The method of claim 1, wherein the compound is4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-2-(4-((R)-2-hydroxypropoxy)-2-fluoro-5-(4,5-dihydro-4-methyl-5-oxotetrazol-1-yl)phenylamino)pyrimindine-5-carbonitrile(Compound 90).
 14. The method of claim 1, wherein the inhibition of thePKC activity results in treatment of a disease or disorder that ismediated or sustained through the PKC activity.
 15. The method of claim14, wherein the disease or disorder is associated with activation of Tcells.
 16. The method of claim 15, wherein the disease or disorder is aninflammatory disease, an autoimmune disease or an ocular disease ordisorder involving inflammatory and/or neovascular events.
 17. A methodof inhibiting a protein kinase C (PKC) activity comprising contacting aPKC with a compound selected from: Compound 88:1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one;and Compound 89:1-(5-(4-((7R,8aS)-5,5-dimethyloctahydroindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-hydroxyphenyl)-4-methyl-1H-tetrazol-5(4H)-one.18. The method of claim 14, wherein the compound is1-(5-(4-((7R,8aS)-octahydro-5,5-dimethylindolizin-7-ylamino)-5-fluoropyrimidin-2-ylamino)-4-fluoro-2-(oxetan-3-yloxy)phenyl)-4-methyl-1H-tetrazol-5(4H)-one(Compound 88).
 19. The method of claim 17, wherein the inhibition of thePKC activity results in treatment of a disease or disorder that ismediated or sustained through the PKC activity.
 20. The method of claim19, wherein the disease or disorder is associated with activation of Tcells.
 21. The method of claim 20, wherein the disease or disorder is aninflammatory disease, an autoimmune disease or an ocular disease ordisorder involving inflammatory and/or neovascular events.